Disruption of the LINC complex to treat laminopathies

Abstract

To provide alternative methods to ameliorate the negative effects overaccumulation of Sun1 has on cells carrying Lmna mutations.SOLUTION: The present invention relates to use of expression vectors and other compounds in methods to disrupt the linker of nucleoskeleton and cytoskeleton (LINC) complex, uncoupling the nucleus from its linkage to the cytoskeleton, resulting in amelioration of diseases caused by one or more Lmna mutations, so-called laminopathies, and more particularly, to the expression of dominant negative SUN domain protein and / or dominant negative KASH domain protein to disrupt, for example, the LINC complex in cardiomyocytes for suppressing disease progression in dilated cardiomyopathy (DCM).SELECTED DRAWING: Figure 19

Description

[Technical Field]

[0001] The present invention relates to the use of expression vectors and other compounds in methods for disrupting the nucleoskeletal-cytoskeletal linker (LINC) complex, dissociating the nucleus from its cytoskeleton, and thereby ameliorating diseases caused by one or more Lmna mutations, so-called laminopathies. More particularly, the present invention relates to the expression of dominant-negative or mutant SUN domain proteins and / or dominant-negative or mutant KASH domain proteins to disrupt the LINC complex in cardiomyocytes, for example, and inhibit disease progression in dilated cardiomyopathy (DCM). [Background technology]

[0002] Dilated cardiomyopathy (DCM) is the most common disease affecting cardiac muscle, accounting for approximately 60% of all cardiomyopathies. DCM is characterized by reduced systolic (contraction) function due to enlargement and thinning of the left ventricular wall, or occasionally both ventricles. DCM is associated with sudden heart failure and cardiac death, leading to high rates of hospitalization, the need for heart transplantation, and consequently high costs [JL Jefferies and JA Towbin, Lancet 375:752-762 (2010); RE Hershberger et al., Nat Rev Cardiol 10:531-547 (2013)]. The causes of DCM are diverse and include a variety of external factors (viruses, autoimmune infiltrates, alcohol, and drugs). However, 30-40% of all cases have a single-gene basis, with mutations in approximately 40 genes being associated with DCM. The most frequently mutated gene in DCM is TTN, which encodes the large sarcomeric protein titin, and truncating variants of TTN account for approximately 15–25% of all congenital forms of DCM [D.S.Herman et al., N Engl J Med 366:619–628 (2012); U.Tayal, S. et al., Genome Med 9:20 (2017)]. The second most frequently mutated gene is lamin A (LMNA), accounting for 6–8% of congenital DCM cases [U.Tayal, S. et al., Genome Med 9:20 (2017)].

[0003] LMNA-induced DCM is characterized by cardiac conduction disease manifested by electrophysiological (ECG) abnormalities, including atrioventricular block, ventricular arrhythmias, and fibrillation. The risk of sudden cardiac death is greater in patients with LMNA cardiomyopathy than in patients with other forms of DCM [JH Van Berio et al., Hum Mol Genet 14:2839-2849 (2005)]. Approximately 450 different mutations have been identified in the LMNA gene, most of which are missense. As a result, the majority of DCM cases are inherited in an autosomal dominant manner. This diversity of mutations complicates genetic approaches to treat LMNA-induced DCM. LMNA-induced DCM can be treated, albeit in limited cases, by adjusting the pacemaker. However, ultimately, effective treatment to date is achieved through heart transplantation (RE Hershberger and A. Morales, in GeneReviews®, edited by M.P. Adam et al. (Seattle, WA, 1993); G. Captur et al., Heart 104:468-479 (2018)).

[0004] Mouse strains carrying Lmna mutations typically die within the first few weeks of life [T. Sullivan et al., J Cell Biol 147:913-920 (1999); A.T. Bertrand et al., Hum Mol Genet 21:1037-1048 (2012); V. Nikolova et al., J Clin Invest 113:357-369 (2004); A.S. Wang et al., Differentiation; research in biological diversity, (2015)]. The cause of early death in mice lacking Lmna is uncertain because multiple tissues are affected. Because Lmna mutant mice develop DCM with conduction abnormalities and focal myocyte degeneration [V. Nikolova et al., J Clin Invest 113:357-369 (2004); L.C. Mounkes et al., Hum Mol Genet 14:2167-2180 (2005)], myocardial damage is likely a major contributing factor, but other, as yet unclear, effects on skeletal muscle may also contribute to early postnatal death.

[0005] Lamins are nuclear intermediate filament proteins and are major components of the nuclear lamina, a protein matrix located inside the inner nuclear membrane (INM). The lamina consists of two major forms, A-type lamins, lamin A and lamin C. A-type lamins are driven by alternative splicing of LMNA, while two B-type lamins (LMNB1 and LMNB2) are encoded by two genes, LMNB1 and LMNB2, respectively [B. Burke and C.L. Stewart, Nat Rev Mol Cell Biol 14:13-24 (2013)]. The lamina confers structural and mechanical integrity to the nucleus, maintains its shape and position within the cell, and is also a determinant of chromatin organization [T. Sullivan et al., J Cell Biol 147:913-920 (1999); I. Solovei et al., Cell 152:584-598 (2013)]. Lamins interact with numerous INM proteins, including emerin, lamina-associated polypeptide (LAP), and SUN domain proteins [B. van Steensel and A.S. Belmont, Cell 169:780-791 (2017)], many of which, when mutated or present as variants, are associated with cardiac disease [H.J. Worman et al., Cold Spring Harbor perspectives in biology 2:a000760 (2010); C.L. Stewart et al., Exp Cell Res 313:2144-2156 (2007)]. Furthermore, these proteins constitute an integrated protein network centered on the lamina, where loss or mutation of lamins can result in mislocalization or altered expression levels of many lamina-associated proteins (emerin, SUN1, LBR, and Lap2α) [T. Sullivan et al., J Cell Biol 147:913-920 (1999); I. Solovei et al., Cell 152:584-598 (2013); C.Y. Chen et al., Cell 149:565-577 (2012); T.V. Cohen et al., Hum Mol Genet 22:2852-2869 (2013); F. Haque et al., J Biol Chem 285:3487-3498 (2010)].Among these proteins whose expression is affected by loss or mutation of Lmna are SUN1 and Lap2, both of which show increased levels. In the case of SUN1, the increased levels are due to reduced turnover rather than increased expression, resulting in high levels accumulating in the Golgi and, at least, Lmna. - / - and LmnaΔ9 mouse disease models [CY Chen et al., Cell 149:565-577 (2012); C. Stewart and B. Burke, WO / 2013 / 158046]. However, when SUN1 levels are genetically reduced in mice carrying the Lmna mutation, this increases lifespan threefold and ameliorates most of the pathology [CY Chen et al., Cell 149:565-577 (2012); C. Stewart and B. Burke, WO / 2013 / 158046]. Wild-type or Sun1 - / - The median survival was over 210 days with a 7-month follow-up period; - / - Mice had a median survival of 41 days; - / - Sun1 + / - Mice had a median survival of 54 days; - / - Sun1 - / - Mice had a median survival of 104 days (Lmna - / - and Lmna - / - Sun1 - / - Similarly, all LmnaΔ9 mice died by 30 days of age, whereas the LmnaΔ9Sun1 mice - / - Littermates survived beyond this period, with most achieving a survival time of more than double this duration [Chen et al., Cell 149:565-577 (2012)]. Hutchison-Gilford Progeria Syndrome Human fibroblasts harboring LMNA mutations that result in LMNA-associated leukemia syndrome (LELS) also exhibit increased Sun1 levels. Deep reduction of Sun1 in these cells alleviated nuclear morphological abnormalities, further suggesting that excess Sun1 caused by LMNA mutations is cytotoxic [C.Y. Chen et al., Cell 149:565-577 (2012); C. Stewart and B. Burke, WO / 2013 / 158046].

[0006] SUN (Sad1p, UNC-84) domain proteins share a conserved C-terminal SUN domain and are localized to the INM [C.J. Malone et al., Development 126:3171-3181 (1999)]. In mammals, SUN1 and SUN2 are the two major SUN proteins widely expressed in virtually all tissues. In the nuclear cisternae between the INM and the outer nuclear membrane (ONM), the C-terminus of SUN1 and / or SUN2 binds to the C-termini (KASH domains) of different Nesprin / SYNE / KASH proteins that cross the ONM. Furthermore, these two protein families constitute the LINC complex, which physically couples the interphase nucleus to the cytoskeleton [M. Crisp et al., J Cell Biol 172:41-53 (2006); E.C. Tapley and D.A. Starr, Curr Opin Cell Biol 25:57-62 (2013)]. The N-terminus of SUN domain proteins protrudes into the nucleoplasm; in SUN1, this region interacts with prelamin A and the nuclear pore complex. It is unclear whether the N-terminus of SUN2 interacts with any nucleoplasmic / NE proteins. In contrast, the majority of Nesprin / KASH domain proteins extend into the cytoplasm adjacent to the ONM. There, depending on the specific Nesprin / KASH protein, they interact directly or indirectly with all three cytoskeletal protein networks (microtubules, actin microfilaments, and intermediate filaments) [HF Horn, Current Topics in Developmental Biology 109:287-321 (2014)]. Furthermore, SUN and KASH / Nesprin proteins of the LINC complex establish a direct physical connection between the cytoplasmic cytoskeletal network (and its connecting components, such as cell adhesion complexes at the plasma membrane) and the interphase interior of the nucleus or the nucleoplasm. The LINC complex is thought to mediate force transmission between the nucleus and the cytoskeleton, thereby controlling changes in gene expression / chromatin organization in response to mechanical / physical stimuli [SGAlam et al., Scientific reports 6:38063(2016)].Loss of either SUN1 or SUN2 alone has no apparent effect on postnatal growth and survival, but SUN1 null mice are sterile and deaf. Simultaneous loss of Sun1 and Sun2 results in perinatal lethality, indicating some degree of redundancy during embryogenesis [K. Lei et al., Proc Natl Acad Sci USA 106:10207-10212 (2009)].

[0007] There is a need to develop alternative methods to ameliorate the negative effects that overaccumulation of Sun1 has on cells carrying Lmna mutations, and the present disclosure aims to provide such methods. Summary of the Invention [Problem to be solved by the invention]

[0008] Surprisingly, the present inventors have found that disruption of the LINC complex, rather than removal of accumulated Sun1 protein, can ameliorate diseases caused by one or more Lmna mutations. One means of achieving disruption is via an expression construct / vector containing an operably linked transgene, where expression of the transgene is via a dominant-negative SUN domain protein or a mutant endogenous SUN domain protein, and / or a dominant-negative KASH domain protein or a mutant endogenous KASH domain protein. This strategy generates a specific KASH domain protein. Exogenous dominant-negative SUN and KASH domain proteins act as LINC complex binding competitors, thereby dissociating the nucleus from its cytoskeleton. Mutant SUN and KASH domain proteins are endogenous Sun and Nesprin proteins that have mutations in the SUN or KASH domain, respectively, and are unable to bind to their cognate LINC complex partners and therefore cannot form the LINC complex. These strategies can be used to disrupt the LINC complex, for example, to treat laminopathies. This result was achieved without actively reducing endogenous SUN1 protein levels. The results presented herein support these claims. [Means for solving the problem]

[0009] According to a first aspect of the present invention, there is provided an isolated nucleic acid molecule comprising an expression vector and a transgene, the transgene being operably linked to the expression vector, and wherein expression of the transgene in a transfected cell results in disruption of a LINC complex in the transfected cell.

[0010] In some embodiments, the expression vector is a cardiac or cardiomyocyte-specific expression vector. In some embodiments, the expression vector comprises a cardiac or cardiomyocyte-specific promoter selected from the group consisting of cardiac troponin T promoter (cTnT), α-myosin heavy chain (α-MHC) promoter, and myosin light chain (MLC2v) promoter. Preferably, the promoter is cardiac troponin T promoter (cTnT).

[0011] In some embodiments, the cardiomyocyte-specific promoter is chicken cardiac troponin T promoter (cTnT). In some embodiments, the expression vector has / is cardiotropic.

[0012] In some embodiments, the expression vector is a viral expression vector. In some embodiments, the viral expression vector is selected from the group including lentivirus, adenovirus, and adeno-associated virus (AAV). Preferably, the viral expression vector is an adeno-associated virus (AAV).

[0013] In some embodiments, the AAV vector is selected from the group consisting of AAV9 (serotype 9), AAV1 (serotype 1), AAV6 (serotype 6), AAV8 (serotype 8), AAV2i8, and AAV9.45.

[0014] In some embodiments, the AAV vector is AAV9 (serotype 9). In some embodiments, the transgene comprises nucleic acid sequences for expressing the luminal domain of a SUN domain-containing protein, an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence.

[0015] In some embodiments, the luminal domain of the SUN domain-containing protein comprises a coiled-coil domain and a SUN domain. In a preferred embodiment, the coiled-coil domain is upstream of the SUN domain.

[0016] In some embodiments, the transgene further comprises nucleic acid sequences for expressing an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence.

[0017] In some embodiments, the transgene comprises an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence, as well as a SUN domain-containing protein. The nucleic acid sequences for expressing either the luminal domain or the SUN domain of the protein are included.

[0018] Preferably, the SUN domain protein is SUN1 or SUN2. In some embodiments, the luminal domain of Sun1 comprises amino acids 458-913 of full-length mouse Sun1 (Uniprot:Q9D666), or its human equivalent, which contains the coiled-coil domain and the SUN domain, but lacks the transmembrane domain. A schematic diagram of the structure of a dominant-negative form of Sun1 is shown in Figure 7.

[0019] For SUN domain constructs, because the SUN domain can bind to the KASH domain, it is expected that the SUN domain alone (crystal structure solved by the Kutay and Schwartz laboratories [Sosa et al., Cell 149(5):1035-47 (2012)], rather than the entire luminal domain (coiled-coil domain and SUN domain), is sufficient to disrupt the SUN-KASH interaction. The human Sun1 SUN domain nucleic acid sequence is set forth in SEQ ID NO: 80. However, the presence of the signal sequence and KDEL sequence is important for targeting the construct to the nuclear cisternae.

[0020] In some embodiments, the N-terminal signal sequence is derived from a secreted protein or a type I transmembrane protein. Preferably, the secretory protein or type I transmembrane protein is selected from the group consisting of human serum albumin, proinsulin, transferrin receptor, EGF receptor, preproopiomelanocortin, pancreatic digestive enzymes (e.g., proteases, amylases, and lipases), endoplasmic reticulum luminal proteins such as protein disulfide isomerase, GRP94, and combinations thereof. More preferably, the N-terminal signal sequence is derived from human serum albumin.

[0021] In some embodiments, the N-terminal signal sequence is not upstream of any other tag at its N-terminus. In some embodiments, the signal peptidase cleavage site is or is derived from one of the group consisting of human serum albumin, proinsulin, transferrin receptor, EGF receptor, prepro-opiomelanocortin, pancreatic digestive enzymes (e.g., proteases, amylases, and lipases), endoplasmic reticulum lumen proteins such as protein disulfide isomerase, GRP94, and combinations thereof. Preferably, the signal peptidase cleavage site is derived from human serum albumin.

[0022] In some embodiments, the C-terminal targeting peptide sequence prevents secretion of the peptide expressed from a transgene according to any aspect of the invention. In some embodiments, the C-terminal targeting peptide sequence is the KDEL tetrapeptide Golgi retrieval sequence. Examples of such structures are shown in Figures 11 and 12.

[0023] In some embodiments, the transgene comprises a humanized Sun1DN or Sun2DN nucleic acid sequence. In a preferred embodiment, the transgene comprises a signal sequence, a humanized Sun1DN nucleic acid sequence, and a KDEL sequence set forth in SEQ ID NO: 4, or the transgene comprises a signal sequence, a humanized Sun2DN nucleic acid sequence, and a KDEL sequence set forth in SEQ ID NO: 5.

[0024] In some embodiments, the transgene further comprises an epitope tag. Preferably, the epitope tag is located at the N-terminus or anywhere in the nucleic acid molecule except downstream (after) the C-terminal targeting peptide sequence [e.g., KDEL], or anywhere in the nucleic acid molecule except upstream (before) the N-terminal signal sequence.

[0025] In some embodiments, the epitope tag is selected from the group consisting of cellulose binding domain (CBD), chloramphenicol acetyltransferase (CAT), dihydrofolate reductase (DHFR), one or more FLAG tags, glutathione S-transferase (GST), green fluorescent protein (GFP), hemagglutinin A (HA), histidine (His), herpes simplex virus (HSV), luciferase, maltose-binding protein (MBP), c-Myc, protein A, protein G, streptavidin, T7, thioredoxin, V5, vesicular stomatitis virus glycoprotein (VSV-G), and combinations thereof. Preferably, the epitope tag is hemagglutinin A (HA).

[0026] In some embodiments, a nucleic acid molecule of the invention comprises an adeno-associated viral vector (AAV) comprising a transgene described in any aspect of the invention comprising the chicken cardiac troponin T promoter (cTnT) and the luminal domain of a SUN domain-containing protein derived from SUN1, an N-terminal signal sequence and signal peptidase cleavage site, each derived from human serum albumin, and a C-terminal targeting peptide sequence that is a KDEL sequence, and the transgene optionally further comprises hemagglutinin (HA) as an N-terminal epitope tag.

[0027] According to one embodiment, an example of such a vector is shown in Figure 10 and comprises the nucleic acid sequence set forth in SEQ ID NO:3. In some embodiments, a nucleic acid molecule of the invention comprises an adeno-associated viral vector (AAV) comprising a chicken cardiac troponin T promoter (cTnT) and a transgene according to any aspect of the invention comprising the luminal domain of a SUN domain-containing protein derived from SUN2, an N-terminal signal sequence and signal peptidase cleavage site, each derived from human serum albumin, and a C-terminal targeting peptide sequence that is a KDEL sequence, and the transgene optionally further comprises hemagglutinin (HA) as an N-terminal epitope tag.

[0028] According to one embodiment, an exemplary nucleic acid molecule may include the vector structure shown in FIG. 10 and the transgene nucleic acid sequence set forth in SEQ ID NO:5. Rather than expressing components of the luminal domain of SUN domain-containing proteins, the KASH domain may be expressed to disrupt the LINC complex by competing with endogenous Nesprin (which contains the KASH domain) for binding to the SUN1 and SUN2 domains.

[0029] Thus, in some embodiments of the nucleic acid molecules of the present invention, the transgene comprises a nucleic acid sequence for expressing a KASH domain and an N-terminal stabilizing polypeptide sequence. Preferably, the KASH domain comprises a transmembrane domain and a SUN-interacting peptide.

[0030] Preferably, the transgene comprises nucleic acid sequences for expressing a KASH domain that spans the nuclear envelope, a SUN-interacting KASH peptide that extends at its C-terminus into the nuclear cisternae, and an N-terminal stabilizing polypeptide sequence in the cytoplasm.

[0031] It will be understood that KASH domain constructs with extensions after the last C-terminal amino acid of a naturally occurring KASH domain are not expected to function, i.e., a C-terminal tag, or even an additional single amino acid at the carboxy terminus, can disrupt the KASH interaction with SUN. In addition, the signal sequence at the N-terminus of the SUN domain construct cannot be upstream of any tag.

[0032] In some embodiments, the KASH domain is selected from the group consisting of KASH1 (derived from Nesprin-1 (SYNE1 gene)), KASH2 (derived from Nesprin-2 (SYNE2 gene)), KASH3 (derived from Nesprin-3 (SYNE3 gene)), KASH4 (derived from Nesprin-4 (SYNE4 gene)), and KASH5 (derived from KASH5 / CCDC155 (KASH5 gene)).

[0033] In a preferred embodiment, the KASH1 domain comprises the human amino acid sequence set forth in SEQ ID NO: 7, the KASH2 domain comprises the human amino acid sequence set forth in SEQ ID NO: 9, the KASH3 domain comprises the human amino acid sequence set forth in SEQ ID NO: 11, the KASH4 domain comprises the human amino acid sequence set forth in SEQ ID NO: 13, and the KASH5 domain comprises the human amino acid sequence set forth in SEQ ID NO: 15. An alignment of the five KASH amino acid sequences is shown in Figure 14.

[0034] In some embodiments, the KASH domain nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the nucleic acid sequence of the KASH1 domain set forth in SEQ ID NO:6, the nucleic acid sequence of the KASH2 domain set forth in SEQ ID NO:8, the nucleic acid sequence of the KASH3 domain set forth in SEQ ID NO:10, the nucleic acid sequence of the KASH4 domain set forth in SEQ ID NO:12, or the nucleic acid sequence of the KASH5 domain set forth in SEQ ID NO:14.

[0035] More preferably, for clinical use, the KASH domain is a human KASH1 domain of SYNE1 having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the nucleic acid sequence of the human KASH1 domain set forth in SEQ ID NO:6.

[0036] It will be understood that due to the redundancy of the genetic code, nucleic acid sequences may have less than 100% identity and still encode the same amino acid sequence. In some embodiments, the KASH domain does not comprise any extension after the last C-terminal amino acid compared to a naturally occurring KASH domain.

[0037] In some embodiments, the N-terminal stabilizing polypeptide sequence is selected from the group consisting of green fluorescent protein (GFP), cellulose binding domain (CBD), chloramphenicol acetyltransferase (CAT), dihydrofolate reductase (DHFR), glutathione S-transferase (GST), luciferase, maltose binding protein (MBP), protein A, protein G, streptavidin, thioredoxin, DHFR, and duplications and combinations thereof.

[0038] In some embodiments, the N-terminal stabilizing polypeptide sequences form separately folded domains. In some embodiments, the vector is an adeno-associated viral vector (AAV) comprising a cardiac troponin T promoter (cTnT), and the transgene comprises a nucleic acid sequence for expressing a KASH domain and an N-terminal stabilizing polypeptide sequence, wherein the KASH domain is selected from the group comprising KASH1, KASH2, KASH3, KASH4, and KASH5.

[0039] In some embodiments, the N-terminal stabilizing polypeptide sequence is green fluorescent protein (GFP). The luminal domain or KASH domain components of SUN domain-containing proteins were expressed to express endogenous Nesprin (containing the KASH domain) or Sun1 and Sun2 ( Another approach to disrupting LINC complexes, rather than disrupting them by competing for binding with endogenous SUN or KASH domains (including SUN domains), is to modify the endogenous SUN or KASH domains so that they are unable to bind to their cognate LINC complex binding partners or have reduced binding affinity for those partners.

[0040] Because both the SUN and KASH domains are located at the C-terminus of their respective proteins, one way to generate modified SUN or KASH domains is to use the CRISPR / Cas system to modify the gene encoding the SUN or KASH domain protein, generating a premature stop codon at the 3' end of the respective protein sequence after CRISPR-induced non-homologous end joining. This can result in truncated proteins with mutated C-terminal SUN or KASH domains. The truncated proteins can be expressed and membrane-localized, but are unable to interact with their cognate LINC complex partners.

[0041] Thus, in some embodiments of the nucleic acid molecules of the present invention, the transgene comprises a nucleic acid sequence for expressing a CRISPR-Cas or other synthetic nuclease system that modifies a nucleic acid encoding the SUN domain of an endogenous Sun protein or the KASH domain of an endogenous Nesprin protein.

[0042] Data presented herein (Example 6) suggest that alterations of the SUN2 or KASH2 domains do not ameliorate Lmna pathology. In some embodiments, CRISPR-Cas modifies the endogenous SUN domain of Sun1 protein or the endogenous KASH domain of Nesprin-1 protein to disrupt the LINC complex. The respective nucleic acids are Sun1 and Syne1.

[0043] In some embodiments, the transgene comprises a nucleic acid sequence for expressing CRISPR-Cas having a gRNA nucleic acid sequence comprising 5'-GCACAATAGCCTCGGATGTCG-3' (SEQ ID NO: 66), which modifies the SUN domain of mouse Sun1.

[0044] In some embodiments, the transgene comprises a nucleic acid sequence for expressing CRISPR-Cas having a gRNA nucleic acid sequence that targets the human SUN1 domain set forth in SEQ ID NO: 80. Preferably, the gRNA nucleic acid sequence targets the end of exon 20, which comprises the nucleic acid sequence set forth in SEQ ID NO: 81. More preferably, the gRNA nucleic acid sequence targets a SUN1 nucleic acid sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, as set forth in Table 3.

[0045] In some embodiments, the transgene comprises a nucleic acid sequence for expressing CRISPR-Cas having a gRNA nucleic acid sequence comprising 5'-CCGTTGGTATATCTGAGCAT-3' (SEQ ID NO: 34), which modifies the KASH domain of mouse Syne-1.

[0046] In some embodiments, the transgene comprises a nucleic acid sequence for expressing CRISPR-Cas having a gRNA nucleic acid sequence that targets the human KASH domain set forth in SEQ ID NO: 6. Preferably, the gRNA nucleic acid sequence comprises a nucleic sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, as set forth in Table 3.

[0047] In some embodiments, the transgene comprises a nucleic acid sequence for expressing CRISPR-Cas9 or a variant thereof. In a preferred embodiment, the transgene is a dominant negative construct.

[0048] In some embodiments, the transgene is a humanized transgene. In some embodiments, expression of the transgene results in disruption of a protein-protein interaction between the SUN domain and the KASH domain of the LINC complex. Preferably, the disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between proteins selected from the group consisting of Sun1+Nesprin-1, Sun2+Nesprin-1, Sun1+Nesprin-2, Sun1+Nesprin-3, Sun2+Nesprin-2, and Sun2+Nesprin-3. More preferably, the disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between proteins with Sun1 and Nesprin-1.

[0049] In some embodiments, the AAV vector is formulated for delivery to the myocardium of a subject. According to a second aspect of the invention, there is provided a nucleic acid molecule of any embodiment of the invention for use in treating a disease caused by one or more Lmna mutations in a subject.

[0050] In some embodiments of the second aspect, the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), mandibuloacral dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and a disease shown in regular font in Table 1.

[0051] [Table 1-1]

[0052] [Table 1-2]

[0053] [Table 1-3]

[0054] [Table 1-4]

[0055] [Table 1-5]

[0056] [Table 1-6]

[0057] [Table 1-7]

[0058] [Table 1-8]

[0059] Table 1-9

[0060] Table 1-10

[0061] Table 1-11

[0062] Table 1-12

[0063] Table 1-13

[0064] Table 1-14

[0065] Table 1-15

[0066] Table 1-16

[0067] Table 1-17

[0068] Table 1-18

[0069] Table 1-19

[0070] Table 1-20

[0071] Table 1-21

[0072] Table 1-22

[0073] Table 1-23

[0074] Table 1-24

[0075] Table 1-25

[0076] Table 1-26

[0077] Table 1-27

[0078] Table 1-28

[0079] Table 1-29

[0080] Table 1-30

[0081] Table 1-31

[0082] Table 1-32

[0083] Table 1-33

[0084] Table 1-34

[0085] Table 1-35

[0086] Table 1-36

[0087] Table 1-37

[0088] Table 1-38

[0089] Table 1-39

[0090] Table 1-40

[0091] Table 1-41

[0092] Table 1-42

[0093] Table 1-43

[0094] Table 1-44

[0095] Table 1-45

[0096] Table 1-46

[0097] Table 1-47

[0098] Table 1-48

[0099] Table 1-49

[0100] Table 1-50

[0101] Table 1-51

[0102] Table 1-52

[0103] Table 1-53

[0104] Table 1-54

[0105] Table 1-55

[0106] Table 1-56

[0107] Table 1-57

[0108] Table 1-58

[0109] Table 1-59

[0110] Table 1-60

[0111] Table 1-61

[0112] Table 1-62

[0113] Table 1-63

[0114] Table 1-64

[0115] Table 1-65

[0116] Table 1-66

[0117] Table 1-67

[0118] Table 1-68

[0119]

Table 1-69

[0120] Table 1-70

[0121] Table 1-71

[0122] Table 1-72

[0123] Table 1-73

[0124] Table 1-74

[0125] Table 1-75

[0126] Table 1-76

[0127] Table 1-77

[0128] Table 1-78

[0129] Table 1-79

[0130] [Table 1-80]

[0131] [Table 1-81]

[0132] [Table 1-82]

[0133] [Table 1-83]

[0134] In some embodiments, the nucleic acid molecule according to any aspect of the invention is for use in treating cardiovascular disease in a subject. In some embodiments, the disease or cardiovascular condition is characterized by the presence of at least one Lmna mutation.

[0135] Preferably, the cardiovascular disease is selected from the group consisting of laminopathies, cardiomyopathies such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, isolated atrial fibrillation; muscular dystrophies (often associated with cardiomyopathy), such as cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, cardiomyopathy associated with congenital muscular dystrophies; premature aging syndromes (which are primarily vascular, but may also have cardiac involvement), such as cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome, and the like, as well as the diseases shown in bold font in Table 1.

[0136] According to a third aspect of the present invention, there is provided an adeno-associated viral vector (AAV) comprising a cardiac troponin T promoter (cTnT) and a transgene according to any aspect of the present invention.

[0137] According to a fourth aspect of the present invention, there is provided a pharmaceutical composition for treating a disease, comprising a nucleic acid molecule according to any embodiment of the present invention. In some embodiments, the disease is a laminopathic disorder.

[0138] In some embodiments, a pharmaceutical composition comprising a nucleic acid molecule according to the invention is for use in treating cardiovascular disease in a subject. According to a fifth aspect of the present invention, there is provided a method of treating a disease in a subject, said method comprising administering a pharmaceutically effective amount of a nucleic acid molecule according to any embodiment of the invention or a pharmaceutical composition of the invention.

[0139] In some embodiments of the methods of treating a disease in a subject, the disease is characterized by the presence of at least one Lmna mutation. In some embodiments of the methods of treating a disease in a subject, the method includes: (i) testing a sample obtained from a subject suspected of having the disease for the presence or absence of at least one Lmna mutation. and the presence of at least one Lmna mutation indicates that the subject should be administered a pharmaceutical composition of the invention or a nucleic acid molecule of the invention.

[0140] In some embodiments of the methods of treating a disease in a subject, the Lmna mutation affects the lamin A or lamin C isoform, or both lamin A / C isoforms of the Lmna gene.

[0141] In some embodiments of the method of treating a disease in a subject, the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), acromandibular dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and a disease shown in regular font in Table 1.

[0142] In some embodiments of the method of treating a disease in a subject, the disease is a cardiovascular disease, and the cardiovascular disease is a laminopathic disorder, a cardiomyopathic disorder, e.g., dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, , dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, isolated atrial fibrillation; muscular dystrophies (often associated with cardiomyopathy), e.g., cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, cardiomyopathy associated with congenital muscular dystrophies; premature aging syndromes (predominantly vascular but may also have cardiac involvement), e.g., cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome; and diseases shown in bold font in Table 1.

[0143] In some embodiments of the methods of treating a disease in a subject, the subject is a non-human mammal, such as a mouse, or a human. In some embodiments of the methods, the mouse is an N195K mouse (Lmna N195K / N195K), or an Lmna conditional knockout (Lmna flox / flox).

[0144] According to a sixth aspect of the invention there is provided the use of a pharmaceutical composition according to the invention or a nucleic acid molecule according to the invention in the manufacture of a medicament for treating a disease caused by one or more Lmna mutations.

[0145] In some embodiments, the disease is an Lmna mutation-associated cardiovascular disease. In some embodiments, the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), acromandibular dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and a disease shown in regular font in Table 1.

[0146] In some embodiments, the cardiovascular disease is selected from the group consisting of laminopathies, cardiomyopathies, such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, and isolated atrial fibrillation; muscular dystrophies (often associated with cardiomyopathy), such as cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, and cardiomyopathy associated with congenital muscular dystrophies; premature aging syndromes (which are thought to be primarily vascular, but may also have cardiac involvement), such as cardiomyopathy associated with atypical Werner syndrome and cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome, as well as diseases shown in bold font in Table 1.

[0147] According to a seventh aspect of the present invention, there is provided a method for screening drug candidates capable of inhibiting or disrupting the LINC complex in a cell. Thus, in some embodiments, there is provided a method for screening drug candidates capable of inhibiting the interaction of proteins of the LINC complex in a cell, comprising: (a) combining proteins of the LINC complex to form a first complex in the presence of a drug; (b) combining the proteins to form a second complex in the absence of the drug; (c) measuring the amount of the first complex and the second complex; and (d) comparing the amount of the first complex with the amount of the second complex. wherein if the amount of the first complex is less than the amount of the second complex, the drug is a drug candidate for inhibiting an interaction of proteins of the LINC complex in a cell.

[0148] In some embodiments, the drug candidate disrupts the protein-protein interaction between SUN and KASH of the LINC complex. Preferably, the drug candidate disrupts the interaction between Sun1 protein and Nesprin-1 protein.

[0149] In some embodiments, the screening is an in vitro screening. In some embodiments, the complex is measured by ELISA. In some embodiments, the recombinant SUN domain is immobilized on a solid surface and the recombinant KASH domain is labeled with an enzyme capable of producing a colorimetric or chemiluminescent readout. Compounds that cannot inhibit the SUN-KASH interaction will result in wells in the plate in which the recombinant SUN can bind to the enzyme-linked KASH domain. After a washing step and incubation with a colorimetric or chemiluminescent enzyme substrate, the presence of the SUN-KASH interaction can be detected in a standard plate reader. If the compound can inhibit the SUN-KASH interaction, the KASH domain will be removed and the enzyme reaction in the well will be reduced or absent after the washing step.

[0150] In some embodiments, if the amount of the first complex is less than the amount of the second complex, the drug is a drug candidate for inhibiting the protein interaction. In some embodiments, the complex is measured by fluorescence anisotropy.

[0151] In some embodiments, fluorescence anisotropy measurements use recombinant SUN and KASH domains. In some embodiments, the KASH domain is fluorescently labeled with a fluorescein moiety, and the fluorescence anisotropy of the KASH domain interacting with the SUN domain can be measured using standard equipment, such as a plate reader incorporating fluorescence spectrometer functionality.

[0152] In some embodiments, if the amount of the first complex is less than the amount of the second complex, there is likely to be a difference in the fluorescence anisotropy of fluorescent KASH, making the drug a candidate drug for inhibiting the protein interaction. [Brief explanation of the drawings]

[0153] [Figure 1] Figure 1 shows a schematic diagram of mutations in the lamin A / C gene, LMNA, and the laminopathies that result from these mutations. [Figure 2] FIG. 2 shows a schematic diagram of the arrangement of components of the nuclear envelope membrane and nuclear lamina. [Figure 3] Figure 3 shows a schematic diagram of the connection between the nucleus and the extracellular matrix via the LINC complex and how mutations in lamin A / C can result in DCM. The plasma membrane, cytoskeleton, and nucleus form a mechanically and physically linked entity. In Lmna mutants, the nucleus is structurally weaker. It is much more susceptible to mechanical stress from cytoskeletal forces. This leads to severe damage to the myocyte nucleus, which initiates a cascade of events, including apoptosis and fibrosis, that ultimately result in DCM. [Figure 4] Figure 4 shows the effect of microinjection of dextran under low pressure into the nuclei of Lmna+ / + and Lmna- / - mice. In wild-type cells, dextran remains in the nucleus, whereas in Lmna mutant cells, dextran effluxes from the nucleus into the cytoplasm. [Figure 5] 5A-5B show a schematic of the LINC complex (FIG. 5A) and the interaction between KASH and SUN (FIG. 5B). [Figure 6] Figure 6 shows that the weight and lifespan defects in Lmna and LmnaΔ9 mice are ameliorated in homozygous Sun1 knockout LmnaSun1 and LmnaΔ9Sun1 animals. (A) Weights are averaged across mice with the indicated genotypes. The number of animals (n) used is indicated. (B) Kaplan-Meier plots show the increased survival of LmnaSun1 compared with Lmna mice. The median survival of wild-type or Sun1 mice was over 210 days over a 7-month follow-up period; LmnaSun1 had a median survival of 41 days; LmnaSun1 had a median survival of 54 days; and LmnaSun1 had a median survival of 104 days (p<0.01 comparing LmnaSun1 with LmnaSun1). (C) Graph showing body weight of LmnaΔ9 mice that are wild-type, heterozygous, or homozygous for Sun1 deficiency. Wild-type and Sun1- / - cohorts are graphed for comparison. Values ​​are the mean ± SEM of animals in each cohort. The number of animals (n) is indicated (p<0.0001 comparing LmnaΔ9Sun1+ / + with LmnaΔ9Sun1- / -). (D) Kaplan-Meier graph showing increased survival of LmnaΔ9Sun1- / - mice compared to LmnaΔ9Sun1+ / + mice. LmnaΔ9Sun1+ / - mice are also graphed (p<0.0001 comparing LmnaΔ9Sun1+ / + with LmnaΔ9Sun1- / -). (E) Graph showing cell proliferation of the indicated MEFs. Curves are the mean ± SD and represent four or more independent isolates from embryos of the indicated genotype. (F) Growth curves of MAFs (mouse adult fibroblasts) from WT, Sun1- / -, LmnaΔ9Sun1+ / +, and LmnaΔ9Sun1- / - mice. MAFs were seeded at a density of 1000 cells per well. Growth was measured, and the normalized cell index (mean ± SD) is shown. [Figure 7]FIG. 7 shows a schematic diagram of the characteristics of the Sun1 protein and the components used to generate dominant-negative SUN1 proteins, including the signal sequence, coiled-coil sequence, SUN domain sequence, and KDEL sequence. [Figure 8] FIG. 8 shows a schematic diagram of the plasmid (SEQ ID NO: 1) used for AAV production. [Figure 9] FIG. 9 shows a schematic diagram of a plasmid for AAV production (SEQ ID NO: 2) containing sequences from AAV2 and AAV9. [Figure 10] FIG. 10 shows a schematic diagram of an AAV expression construct (SEQ ID NO: 3) containing a cardiac-specific promoter and a Sun1 dominant-negative sequence. [Figure 11] FIG. 11 shows a schematic diagram of the characteristics of the dominant-negative Sun1 protein, including the signal sequence, the coiled-coil sequence, the SUN domain sequence, and the KDEL sequence (SEQ ID NO: 4). [Figure 12] FIG. 12 shows a schematic diagram of the characteristics of the dominant-negative Sun2 protein, including the signal sequence, the luminal domain sequence, and the KDEL sequence (SEQ ID NO: 5). [Figure 13] FIG. 13 shows a schematic diagram of the regions of the Sun1 protein used in the dominant-negative constructs. [Figure 14] Figure 14 shows an alignment of the KASH1 to KASH5 domain amino acid sequences (SEQ ID NOs: 7, 9, 11, 13, and 15, respectively) with conserved residues. [Figure 15]Figure 15 shows schematic diagrams of the LINC complex in wild-type mice, Sun1 KO mice, AAV dominant-negative SUN mice, and mice with altered KASH domains. The schematic diagram of the wild-type mice is taken from Brian Burke, 2012. The schematic diagram of the Sun1 KO mice represents the results of Chen et al., 2012. The schematic diagrams of the AAV dominant-negative SUN domain and altered KASH domain represent the inventors' submission at the priority date regarding methods for disrupting the LINC complex to ameliorate laminopathies and are based on data obtained at that time. [Figure 16] FIG. 16 shows Kaplan-Meier curves for Lmna KO mice surviving an average of 28 days, Sun1 KO mice living for more than 300 days, and cardiac Lmna KO / Sun1 KO mice living for more than 300 days. [Figure 17] FIG. 17 shows H&E stained sections of hearts from Sun1 KO mice, cardiac Lmna KO mice, and cardiac Lmna KO / Sun1 KO mice, with LmnaKO / Sun1WT hearts showing left ventricular enlargement (DCM) compared to WT and LmnaKo / Sun1KO hearts. [Figure 18] Figure 18 shows a schematic diagram of LINC complex disruption in Nesprin-1 ΔKASH mice. LmnaKO Nesprin-1WT mice have a survival time of approximately 20 days. LmnaKO Nesprin-1-ΔKASH mice survive for approximately 40 days, comparable to LmnaKOSun1KO mice. [Figure 19] Figures 19A-19B show a schematic diagram of expected AAV-cTNT-DN-SUN expression, as well as the competition between exogenous DN-SUN and native Sun1 for binding to the KASH domain (Figure 19A), as shown in 19B (top panel), and the effect of transfected DN-SUN on the localization of native Nesprin2G in cells in which two nuclei express DN-SUN in the middle panel and both show loss of Nesprin2 from the nuclear membrane in the merge panel (Figure 19B). [Figure 20]Figure 20 is a Kaplan-Meier curve showing that in vivo disruption of the SUN-KASH interaction using AAV9-cTNT dominant-negative Sun1 (DNSun1) extends the lifespan of heart-specific Lmna KO in male and female mice. [Figure 21] Figure 21 shows the C-terminal amino acids of the KASH domain of Nesprin-2 (KASH2). A sequence of 14 or 18 amino acids from the KASH2 C-terminus can physically interact with the SUN domain of SUN2. Loss of the last four amino acids from KASH2 or addition of a single alanine amino acid at the C-terminus of KASH2 is sufficient to disrupt the interaction between the KASH2 domain and the SUN domain. [Figure 22] FIG. 22 shows a schematic diagram of a screening method for detecting agents that disrupt LINC complexes. [Figure 23] FIG. 23 shows a flow chart illustrating a more detailed screening method for identifying small molecules that disrupt LINC complexes. [Figure 24] Figure 24 shows Kaplan-Meier curves showing that wild-type (C57 / Bl6) mice with or without the Nesprin-1 KASH-disrupting (C'TΔ8) mutation have a normal survival time. Mice wild-type (Nesp1+ / +) or heterozygous for Nesp1-C'TΔ8 (Nesp1+ / C'TΔ8) with the Lmna null / KO mutation (LA-ZP3creΔ / Δ) have a median survival time of 15 or 18 days, while median survival time increases to 38 days in Lmna KO / Nesp1 homozygous (LA-ZP3creΔ / Δ; Nesp1C'TΔ8 / C'TΔ8) mice. [Figure 25]Figure 25 shows Kaplan-Meier curves showing that mice with wild-type Lmna (N1CTΔ8 / CTΔ8LA+ / +MCre+ / -) or mice with a loxP-transfected Lmna allele but lacking the cardiac-specific Cre driver (N1CTΔ8 / CTΔ8LAf / fMCre+ / + and N1WT / WTLAf / fMCre+ / +) survive for the duration of the experiment (approximately 80 days at the time of priority filing, extending unchanged to 120 days). Mice with cardiomyocyte-specific deletion of Lmna (N1WT / WTLAf / fMCre+ / -) have a survival time of 22-24 days after induction of Cre / loxP-mediated deletion by tamoxifen (TMX) delivery, and survival time increases up to the duration of the experiment in mice with TMX-induced cardiomyocyte-specific deletion of Lmna and a homozygous mutation for Nesprin-1 (N1CTΔ8 / CTΔ8LAf / fMCre+ / -). [Figure 26-1] Figures 26A-26D show Kaplan-Meier curves for the lifespan extension of Lmna mutant mice due to Sun1 loss. (Figure 26A) Wild-type (C57 / Bl6) mice with or without Sun1 had normal lifespans, whereas LmnaFlx / Flx:Zp3 mice, in which lamin A is deleted in all tissues, had a mean postnatal survival of 17.5 days (***P ≤ 0.0001, log-rank test). On the Sun1- / - background, lifespan increased to 32.5 days. (Figure 26B) When LmnaFlx / Flx mice were specifically and constitutively deleted in the heart by crossing them with the CreαMyHC line, LmnaFlx / Flx:αMyHC mice lived an average of 26.5 days. On the Sun1- / - background, these mice lived for more than 6 months. [Figure 26-2](Figure 26C) When 3- to 5-month-old LmnaFlx / Flx mice were crossed with Tmx-inducible cardiomyocyte-specific Cre Tg{Myh6-cre / Esr1) (abbreviated as mcm), the mice died within 3-4 weeks after a single injection of Tmx. On the Sun1- / - background, these mice lived for over 1 year. (Figure 26D) Compared with LmnaN195K / N195KSun1- / - mice, which had a mean survival time of 111 days, LmnaN195K / N195K mice lived an average of 78 days. (***P ≤ 0.0001, **P = 0.0073, log-rank test). [Figure 27-1] Figures 27A-27E show the survival and phenotype of LmnaFlx / Flx:mcm + Tmx mice. (Figure 27A) The mean survival time of LmnaFlx / Flx:mcm mice was 27 days after a single Tmx injection (***P≦0.0001, log-rank test). (Figure 27B) PCR detected the loxP-transduced (deleted) Lmna gene (arrowhead) only in cardiac tissue after Tmx injection, but not in other tissues, even when Tmx was not injected. [Figure 27-2] (FIG. 27C) LmnaFlx / Flx:mcm+Tmx mice developed kyphosis (arrowhead markings) by 21 days post-injection. [Figure 27-3] (Figure 27D) Cardiomyocyte (CM) nuclei were detected by PCM-1 staining 21 days after Tmx. Lamin A / C protein, detected by immunofluorescence, was present in controls (i, iii) but was reduced / absent (white arrowhead) in CM nuclei of both isolated CMs (second panel of ii) and heart sections (iv). [Figure 27-4](Figure 27E) Lamin A / C levels were quantified by Western analysis of whole heart lysates 21 days after injection. A significant decrease in A-type lamin protein (***P≦0.0001, T-test) was detected, but lamin C levels were not significantly reduced in LmnaFlx / Flx:mcm+Tmx mice compared with LmnaFlx / Flx:mcm+CTL. (Figure 27F) Quantitative analysis was performed 21 days after Tmx. The presence of LoxP sites in the WT-Lmna gene (LmnaFlx / Flx) resulted in reduced Lmna transcript levels compared with LmnaWt / Wt levels, but this had no apparent effect on lifespan or growth / survival. [Figure 28-1] Figures 28A-28D show echocardiograms, cardiac function, and histology of LmnaFlx / Flxmcm+Tmx mice. (Figure 28A) LmnaFlx / Flx:mcm+Tmx mice exhibit reduced cardiac contractile function. (Figure 28B) LmnaFlx / Flx:mcm hearts exhibit reduced EF% and FS% and increased LVID (***P≦0.0001, **P=0.0010, two-way ANOVA). [Figure 28-2] (Figure 28C) Histological analysis of the heart revealed increased infiltration of nucleated cells and intercellular spaces in LmnaFlx / Flx:mcm hearts (i and ii). Significantly fewer viable (brick-like) CMs were isolated from LmnaFlx / Flx:mcm hearts compared with LmnaFlx / Flx:mcm controls (iii). Further magnification revealed that cardiomyocytes isolated from LmnaFlx / Flx:mcm hearts contained large intracellular vacuoles (arrowhead, iv). [Figure 28-3] (Fig. 28D) The left ventricular lumen in LmnaFlx / Flx:mcm hearts was enlarged (i), which was accompanied by increased fibrosis (ii) (**P=0.0007, seen as the light gray area in the middle panel ii of 28D and the left panel of iv) and an increase in apoptotic nuclei as revealed by TUNEL staining (*P=0.0220, one-way ANOVA) (right panels of iii and iv). All sampling and analysis was performed on hearts 21 days after Tmx injection. [Figure 29-1]Figures 29A-29D show changes in nuclear morphology and cardiac structure in LmnaFlx / Flx:mcm mice with or without Sun1 after Tmx injection. (Figure 29A) CM nuclei with reduced or absent lamin A / C expression are indicated by white arrowheads (1, 3). CM nuclei with normal lamin A / C levels (2, 4) are indicated by gray arrowheads. [Figure 29-2] LMNA protein levels, measured by both fluorescence intensity (5) and Western blot (6), were significantly reduced in LmnaFlx / Flx:mcmSun1+ / ++Tmx (***P = 0.0009, T-test) and LmnaFlx / Flx:mcmSun1- / -+Tmx (*P = 0.0359, T-test) compared with LmnaFlx / FlxmcmSun1+ / + controls (lower graph, 6). [Figure 29-3] (FIG. 29B) Left ventricular (LV) enlargement was evident in LmnaFlx / Flx:mcmSun1+ / ++Tmx hearts (panel 1), but not in the LV of LmnaFlx / Flx:mcmSun1- / -+Tmx hearts (panel 2). LmnaFlx / Flx:mcmSun1+ / ++Tmx mice had significantly increased fibrosis compared with controls (panel 3, fibrosis is gray), whereas there was no significant increase in fibrosis in LmnaFlx / FlxmcmSun1- / -+Tmx hearts (panel 3) compared with controls (panels 4, quantified in panel 5; ***P=0.0001, one-way ANOVA). Cardiac papillary muscle force measurements from LmnaFlx / Flx:mcmSun1+ / ++Tmx mice were significantly reduced compared to LmnaFlx / Flx:mcmSun1+ / + controls (**P=0.0047, T-test) and LmnaFlx / Flx:mcmSun1- / -+Tmx (*P=0.0113, T-test) (Panel 6). [Figure 29-4](Figure 29C) CM nuclear morphology was significantly altered in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice (panel 1, filled arrowheads). In the absence of TMX, control heart sections (CTL, panel 2) display a few nuclear abnormalities. In the absence of Sun1, LmnaFlx / Flx:mcmSun1- / -+Tmx cardiomyocytes showed no nuclear abnormalities (panels 3 and 4). Panel 5 of Figure 29C reveals that, in summary, 70% of CMs in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice had ruptured / distorted NEs or malformed nuclei, compared with less than 1% of CM nuclei in LmnaFlx / Flx:mcmSun1- / -+Tmx mice. [Figure 29-5] (Figure 29D) Echocardiogram analysis was performed on TMX-treated and control mice after Tmx induction. Echocardiograms (ECGs) performed 28 days after Tmx injection on 3- to 5-month-old mice (Panel 1). ECGs performed before and after Cre induction revealed a progressive deterioration of cardiac contractility in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice (solid black line) compared with LmnaFlx / Flx:mcmSun1- / -+Tmx mice (Panels 2 to 4). Loss of SUN1 maintained EF (Panel 2), FS (Panel 3), and longitudinal global strain (GLS, Panel 4) in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice compared with LmnaFlx / Flx:mcmSun1+ / ++Tmx mice. [Figure 30]Figures 30A-30B show Kaplan-Meier plots of SUN1 deletion on cardiac pathology induced by a missense mutation (N195K) in the Lmna gene and the effect of SUN1 deletion on cardiac function. (Figure 30A) Absence of Sun1 significantly increases the survival time of LmnaN195K / Flx:mcmSun1- / -+Tmx mice compared with LmnaN195K / Flx:mcmSun1+ / ++Tmx mice (*P=0.0101, log-rank test). Mice with only one copy of the N195K mutation (LmnaN195K / -:mcmSun1+ / ++Tmx) had a median survival time of 47 days, approximately half the survival time of homozygous mice with two copies of the N195K allele. (Figure 30B) Echocardiograms (ECGs) performed before and after Cre induction revealed a progressive deterioration of cardiac contractility in LmnaN195K / -:mcmSun1+ / ++Tmx mice over time compared with LmnaN195K / -:mcmSun1- / -+Tmx mice. ECG images were recorded 28 days after Tmx injection (left-hand panel). Loss of SUN1 preserved EF, FS, and GLS in LmnaN195K / Flx:mcmSun1- / -+Tmx mice compared with LmnaN195K / Flx:mcmSun1+ / ++Tmx mice (lower three right-hand panel). [Figure 31-1]Figures 31A-31G show that LmnaFlxx / Flx:mcm+Tmx mice expressing AAV-transduced DNSun1 exhibit improved cardiac function and increased lifespan. (Figure 31A) A diagram showing the protocol for AAV-mediated transduction of DN-Sun1 miniprotein into LmnaFlxx / Flx:mcm+Tmx mice. A single IP Tmx injection is administered on postnatal day 14 to induce Lmna deletion. AAV9-DNSun1 or AAV9-GFP viral particles are then injected into the thoracic cavity on postnatal day 15. The experimental endpoint was set at 100 days after Tmx. (Figure 31B) The DNSun1 miniprotein competes with endogenous Sun1 for binding to the KASH domain of Nesprin (which is Nesprin1 in CM). The miniprotein competes with endogenous Sun1 for binding to the KASH domain of Nesprin. Because the DNSun1 miniprotein does not anchor in the INM, it effectively displaces endogenous SUN proteins from binding to the KASH domain, thus disrupting the LINC. (Figure 31C) The presence of the recombinant Lmna gene after Tmx injection was confirmed by PCR in cardiac tissue (upper panel). Strong expression of both AAV9-DNSun1 and AAV9-GFP proteins (dosage: 5 × 10^10 vg / g mouse) was detected in extracts from whole hearts 99 days after AAV injection (lower panel). (Figure 31D) CM derived from human iPS cells were transduced with DNSun1 using AVV-DJ as a vector. In CMs expressing high levels of DNSun1, as indicated by gray arrows, Nesprin1 localization to the NE was reduced or absent. Nesprin1 localization to the NE was maintained in CMs that either did not express or expressed low levels of AVV-DJ-DNSun1 (white arrowhead). [Figure 31-2](Figure 31E) LmnaFlx / Flx:mcm+Tmx+AAV9-GFP mice lived an average of 34.5 days after Tmx induction, whereas LmnaFlx / Flx:mcm+Tmx mice injected with AA9-DNSun1 (5 x 10^10 vg / g / mouse) lived significantly longer (**P = 0.0038, log-rank test) until at least 100 days after Tmx, at which point they were sacrificed for analysis. This data set was derived from that shown in Figure 20 and adjusted by removing female mice and mice treated with different doses of virus. Panel E(i) represents male mice, and Panel E(ii) represents female mice. [Figure 31-3] (Fig. 31F) At 35 days after Tmx, extensive fibrosis (blue in the original image, gray here) and ventricular enlargement were detected in LmnaFlx / Flx:mcm+Tmx+AAV9-GFP hearts compared with LmnaFlx / Flx:mcm+Tmx+AAV9-DNSun1 hearts. (Fig. 31G) ECG analysis confirmed that LmnaFlx / Flx:mcm+Tmx+AAV9-DNSun1 hearts had better cardiac function than LmnaFlx / Flx:mcm+Tmx+AAV9-GFP hearts at 35 days after Tmx injection. [Figure 32] Figures 32A-32D show a model of how disrupting LINCs by disrupting Sun1 protects cardiomyocytes from contraction-induced stress. (Figure 32A) Cardiomyocyte nuclei expressing LmnaA / C can withstand mechanical stress and tension transmitted from the cytoplasm to the NE via the LINC complex. (Figure 32B) Loss of the Lmna gene or introduction of a mutation within the Lmna gene results in the loss and / or incorrect assembly of the nuclear lamina, weakening the lamina / NE. The weakened nuclei are damaged by tension / stress exerted by the contracting sarcomeres of cardiomyocytes via the LINC complex. (Figures 32C, D) In ​​the absence of SUN1 or by disrupting the binding of SUN1 to the KASH domain by expressing DNSun1, the untethered LINC complex exerts less tension on the cardiomyocyte nucleus, allowing Lmna mutant cardiomyocytes to survive. [Figure 33]Figure 33 shows the structure of the LmnaFlx / Flx conditional allele. Primer positions for identifying the genotype of the Lmna gene both before and after Cre recombination are shown for the LmnaFlx allele (Flox), the Lmna deletion allele (Δ), and the wild-type allele [AS Wang et al., Differentiation 89:11-21 (2015)]. [Figure 34] Figure 34 shows a diagram of the recombinant AAV9-DNSun1 and AAV9-GFP miniproteins. DN-Sun1 contains the SUN domain, an HA tag, a signal sequence (SS, for targeting the protein to the ER), and KDEL (an ER retention signal) [M. Crisp et al., J Cell Biol. 172:41-53 (2006)]. AAV9-GFP contains the SS and KDEL sequences. GFP was used instead of Sun1L-KDEL as a control. [Figure 35] Figure 35 shows a photomicrograph of cardiomyocyte-specific expression of Cre recombinase after Tmx injection. LmnaFlx / Flx:mcm mice were crossed with mT / mG (JAX:Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J) reporter mice. In the absence of Cre, RFP is expressed. When Cre is induced, GFP is expressed. Only CMs of LmnaFlx / Flx:mcm mice express GFP upon TMX injection. Heart tissue was analyzed 7 days after Tmx injection. [Figure 36] Figure 36 shows that loss of Sun2 does not rescue loss of Lmna. Loss of Sun2 does not extend the survival of LmnaΔ / ΔSun2− / − mice. [Figure 37-1] Figures 37A-37E show the phenotypes of LmnaFlx / FlxmcmSun1+ / + and LmnaFlx / FlxmcmSun1- / - hearts 12-14 months after Tmx injection. (Figure 37A) Histological analysis of senescent LmnaFlx / Flx / :mcmSun1+ / + hearts 12-14 months after Tmx injection reveals no significant morphological changes compared to controls, such as LV enlargement or (Figure 37B) fibrotic changes. [Figure 37-2]Figures 37A-37E show the phenotypes of LmnaFlx / FlxmcmSun1 and LmnaFlx / FlxmcmSun1 hearts 12-14 months after Tmx injection. (Figure 37A) Histological analysis of senescent LmnaFlx / Flx / :mcmSun1 hearts 12-14 months after Tmx injection reveals no significant morphological changes compared to controls, such as LV enlargement or (Figure 37B) fibrosis. (Figure 37C) PCR analysis confirmed the sustained deletion of the Lmna gene. [Figure 37-3] (FIG. 37D) Protein quantification revealed a significant decrease in LMNA levels in LmnaFlx / Flx:mcmSun1− / −+Tmx hearts 14 months after TMX. [Figure 37-4] (FIG. 37E) Echocardiograms from aged mice (left-hand panel) showed decreased EF and FS (right-hand panel) in both LmnaFlx / Flx:mcmSun1+ / ++CTL aged mice and LmnaFlx / Flx:mcmSun1− / −+Tmx aged mice. [Figure 38] Figure 38 shows that rescue by AAV9-DNSun1 depends on the dosage of injected viral particles. Survival of LmnaFlx / Flx:mcm+TMX mice depends on the dosage of AAV9-DNSun1, with lower concentrations resulting in shorter survival. Each point represents a mouse, and the horizontal line indicates the mean. [Figure 39-1] Figures 39A-39C show the levels of lamin A / C and the expression of AAV-expressed proteins after Tmx induction. (Figure 39A) Lamin A / C levels were significantly reduced after Tmx induction, and the presence of either AAV9-DNSun1 or AAV9-GFP proteins did not alter LMNA protein levels (quantification of lamin A / C immunofluorescence intensity). The amounts of lamin A / C, DNSun1, and GFP proteins in whole hearts were also quantified by Western analysis (bottom three graphs). (Analysis performed 35 days after Tmx.) [Figure 39-2](Figure 39B) Expression of both DNSun1 and GFP proteins depended on the concentration of injected viral particles. (Figure 39C) Immunofluorescence revealed that the majority of CMs were successfully infected and expressed GFP with 5x10^10 vg / g AAV9-GFP (left image) compared with infection with a ten-fold lower concentration of viral particles (5x10^9 AAV9-GFP, right image). [Figure 40-1] Figures 40A-40C show that targeting the Sun1 SUN domain with CRISPR results in loss of Sun1 protein. (A, B) Clustal alignment of Sun1 DNA (Figure 40A) and amino acid (Figure 40B) sequences flanking CRISPR-induced mutations in wild-type Sun1 (SEQ ID NOs: 69 and 72, respectively), Sun1 with a 4-bp insertion (Sun1_plus4; SEQ ID NOs: 70 and 73, respectively), and Sun1 with a 7-bp deletion (Sun1_del7; SEQ ID NOs: 71 and 74, respectively). Numbering is for the Sun1 coding sequence (A) and protein sequence (B). Bold text in (B) indicates the SUN domain. [Figure 40-2] Figures 40A-40C show that targeting the Sun1 SUN domain with CRISPR results in loss of Sun1 protein. (A, B) Clustal alignment of Sun1 DNA (Figure 40A) and amino acid (Figure 40B) sequences flanking CRISPR-induced mutations in wild-type Sun1 (SEQ ID NOs: 69 and 72, respectively), Sun1 with a 4-bp insertion (Sun1_plus4; SEQ ID NOs: 70 and 73, respectively), and Sun1 with a 7-bp deletion (Sun1_del7; SEQ ID NOs: 71 and 74, respectively). Numbering is for the Sun1 coding sequence (A) and protein sequence (B). Bold text in (B) indicates the SUN domain. [Figure 40-3] (FIG. 40C) Immunofluorescence staining of adult mouse fibroblasts derived from wild-type and Sun1 mutant mice. Sun1 expression is lost in mutant mice, while Sun2 and Nesprin-1 expression is similar in all three genotypes. Scale bar = 10 μm. [Figure 41-1]Figures 41A-D show that targeting the Syne1 C-terminus with CRISPR results in the expression of mutant Nesprin-1 proteins. (A, B) Clustal alignment of wild-type Nesprin-1 DNA (SEQ ID NO: 75) and Nesprin-1C'TΔ8 (Nesprin1_CTdel8) (SEQ ID NO: 76) (A) and the amino acid sequences flanking the CRISPR-induced mutations in wild-type Nesprin-1 (SEQ ID NO: 77) and Nesprin-1C'TΔ8 (Nesprin1_CTdel8) (SEQ ID NO: 78) (B). Bold TGA indicates the stop codon of the Syne1 / Nesprin-1 gene. [Figure 41-2] (C, D) Immunoblot of Nesprin-1 from Syne1 / Nesprin-1 wild-type and Syne1 / Nesprin-1C'TΔ8 mutant heart and muscle tissues. [Figure 42] Figures 42A-B are photomicrographs showing that CRISPR-induced Syne1 mutation results in mislocalized "KASH-free" Nesprin-1 protein. Immunofluorescence staining of mouse adult fibroblasts (A) and primary myotubes (B) derived from wild-type (WT) and Syne1C'TΔ8 mutant mice. Nesprin-1 is mislocalized from the nuclear envelope in mutant samples. Merged images show Nesprin-1 and DNA staining. Scale bar = 10 μm. [Figure 43] Figure 43A-C are photomicrographs showing that Syne1 mutations do not disrupt the localization of certain nuclear envelope proteins. (A-C) Immunofluorescence staining of mouse primary myotubes derived from wild-type (WT) and Syne1C'TΔ8 mutant mice. Sun1 (A), Sun2 and emerin (B), and lamin A / C (C) normally localize to the nuclear envelope. Merged images show protein and DNA staining. Arrows indicate examples of normally localized nuclear envelope proteins. Scale bar = 10 μm. [Figure 44]Figure 44A-C are photomicrographs showing that Syne1 mutation disrupts the localization of nuclear envelope-localized centrosomal proteins. (A-C) Immunofluorescence staining of mouse primary myotubes derived from wild-type (WT) and Syne1C'TΔ8 mutant mice. Pcm1, pericentrin (Pcnt), and Akap450, which normally localize to the nuclear envelope in myotubes, are displaced from the nuclear envelope in Syne1C'TΔ8 mutant myotubes. MF20 is an antibody directed against myosin heavy chain, a myotube marker. Merged images show protein and DNA staining. Arrows indicate typical nuclear envelope staining for these centrosomal proteins. Scale bar = 10 μm. [Figure 45] Figures 45A-C show that Syne1 mutations do not affect mouse phenotype. (A-B) Exemplary images of 12-week-old male (A) and female (B) mice. (C) Body weights of male and female wild-type (WT) and Syne1C'TΔ8 mutant mice over a 6-week period. [Figure 46] Figures 46A-C show that Syne2 constructs and Syne1 / Syne2 double mutant mice experience perinatal lethality. (Figure 46A) Design of the IRES-βgal PGK-Neo targeting construct to generate Syne2 mutations. (Figure 46B) Immunofluorescence staining of adult mouse fibroblasts derived from wild-type (WT) and Syne2 mutant mice showing loss of Nesprin-2. (Figure 46C) Images of newborn pups. The top row shows healthy, pink-appearing mice with at least one wild-type Syne1 or Syne2 allele. The bottom row shows cyanotic, double-mutant Syne1C'TΔ8 / C'TΔ8:syne2- / - pups that appear blue and die at birth. [Figure 47] Figure 47 is a Kaplan-Meier graph showing that Syne2 mutation does not ameliorate Lmna pathology. Kaplan-Meier survival curve showing that LmnaΔ / Δ mice die within 3 weeks of birth, regardless of Syne2 mutation status (wild-type, heterozygous, or mutant). DETAILED DESCRIPTION OF THE INVENTION

[0154] definition Certain terms employed in the specification, examples, and appended claims are collected here for convenience.

[0155] The term "amino acid" or "amino acid sequence," as used herein, refers to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. When "amino acid sequence" is used herein to refer to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms are not meant to limit the amino acid sequence to the complete, naturally occurring amino acid sequence associated with the described protein molecule.

[0156] As used herein, the term "comprising" or "including" should be interpreted as specifying the presence of the stated features, integers, steps, or components as stated, but does not exclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. However, in the context of the present disclosure, the term "comprising" or "including" also includes "consisting of." Conjugations of the word "comprising," such as "comprise" and "comprises," and conjugations of the word "including," such as "include" and "includes," have correspondingly varied meanings.

[0157] As used herein, the terms "CRISPR-Cas" and "CRISPR" system are used somewhat interchangeably and refer to a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. It comprises clustered regularly interspaced short palindromic repeats (CRISPR), a CRISPR-associated (Cas) endonuclease, and a synthetic guide RNA that can be programmed to identify and introduce double-strand breaks at specific sites within the targeted genetic sequence. The palindromic repeats are spaced apart by short, variable sequences derived from the exogenous DNA target, known as protospacers, which together comprise the CRISPR RNA (crRNA) array. Within the DNA target, each protospacer is always associated with a protospacer adjacent motif (PAM), which can vary depending on the specific CRISPR system. CRISPR-Cas9 is a novel CRISPR-based system that utilizes R This is a specific mode of the system, involving the use of the Cas9 nuclease guided by NA, originally derived from Streptococcus pyogenes, which requires that the target DNA be located immediately before the 5'-NGG PAM. Variants of the CRISPR-Cas9 system, including CRISPR-Cpf1, are known [Ran FA et al., Nat. Protoc 8, 2281-2308 (2013); Ran FA et al., Cell 154, 1380-1389 (2013)], and although CRISPR-Cas9 is used in the examples herein, it is not intended that the present invention be limited to a particular CRISPR-Cas system.

[0158] As used herein, the term "dominant negative" refers to a mutation in which a gene product adversely affects the normal wild-type gene product in the same cell.This usually occurs when the product can still interact with the same elements as the wild-type product, but blocks some aspects of its function.In one example, a transgene is expressed as a protein that is functional as a dimer.A mutation that removes a functional domain but retains a dimerization domain can cause a dominant negative phenotype, because some part of the protein dimer may lack one of the functional domains.

[0159] As used herein, the term "regular font" in connection with a disease listed in Table 1 refers to a disease that is written in plain type and not in bold type. The term "bold type" has its general meaning.

[0160] As used herein, the term "stabilizing polypeptide" or "stabilizing protein" refers to an inactive polypeptide that folds into separate domains, thereby ensuring that the remainder of the peptide maintains, for example, the proper topology. In one example, a stabilizing protein ensures that KASH proteins maintain the proper topology on the endoplasmic reticulum membrane and the nuclear envelope. In another example, a stabilizing polypeptide prevents the attached polypeptide from translocating, for example, to the nuclear cisternae.

[0161] As used herein, the term "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their specific functions under suitable conditions. For example, a control sequence "operably linked" to a protein-coding sequence is ligated to the protein-coding sequence such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. By way of example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when it is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of that coding sequence. Overall, operably linked DNA sequences are contiguous and, where necessary to link two protein-coding regions, in the same reading frame.

[0162] As used herein, the term "extension" refers to one or more amino acids that may be found attached to the N- or C-terminus of a desired peptide. As used herein, the terms "polypeptide," "peptide," or "protein" refer to one or more chains of amino acids, where each chain comprises amino acids covalently linked by peptide bonds; the polypeptide or peptide can comprise multiple chains noncovalently and / or covalently linked together by peptide bonds, having the sequence of a native protein, i.e., a protein occurring in nature and produced by a non-specifically engineered cell, or a protein produced by a genetically engineered or recombinant cell, and can constitute a molecule having the amino acid sequence of a native protein or a molecule having one or more amino acid deletions, additions, and / or substitutions of such amino acids from the native sequence. A "polypeptide," "peptide," or "protein" can comprise one (referred to as a "monomer") or multiple (referred to as a "multimer") amino acid chains.

[0163] The term "subject" is defined herein as a vertebrate, particularly a mammal, more particularly a human. For research purposes, the subject may particularly be at least one animal model, such as a mouse and a rat. In particular, for the treatment or prevention of a laminopathic disorder such as DCM, the subject may be a human.

[0164] The term "treatment" as used in the context of the present invention refers to restorative, therapeutic or curative treatment. Without being bound by theory, the inventors believe that the entire therapeutic principle is to suppress Lmna mutations by disrupting LINC complex function. It is further noted that the target of the claimed method is the SUN-KASH interaction in the LINC complex. Endogenous protein levels should not be affected.

[0165] It is further noted that if a full-length SUN domain protein is inserted between the signal sequence and KDEL, the transgene (e.g., a dominant-negative transgene) may not function because the SUN domain inverts the membrane topology of the protein so that it is no longer located in the nuclear cisterna / ER lumen. Only the region following the transmembrane domain, i.e., the luminal domain, may be used.

[0166] Those skilled in the art will understand that the present invention can be practiced according to the methods described herein without undue experimentation. The methods, techniques, and chemicals are described in the cited references or are from protocols in standard textbooks of biotechnology and molecular biology. [Example]

[0167] Example 1 material and method Mice were maintained at the A*STAR Biological Resource Centre facility and the NUS Animal Facility in accordance with the guidelines of each institution's Animal Care and Use Committee. Flx / Flx Mice were generated and characterized as previously described [A.S. Wang et al., Differentiation; research in biological diversity, (2015); I. Solovei et al., Cell 152:584-598 (2013)] (Figure 33). Δ / Δ In order to obtain mice carrying the loxP sequence, a loxP sequence was introduced into the allele (Lmna Flx / Flx ) was introduced by crossing into mice carrying the Cre recombinase driven by the regulatory sequences of the mouse zona pellucida 3 gene (Zp3; Tg(Zp3-cre)93Knw, JAX stock 003651) [W. de Vries et al., Genesis 26:110-112 (2000)]. Flx / Flx / NIMhc ) to get Lmna Flx / FlxMice were crossed to mice in which Cre expression was driven by the cardiac-specific mouse alpha myosin heavy chain (Myh6, myosin, heavy polypeptide 6, cardiac muscle, alpha) promoter (MyHC; Tg(Myhca-cre)2182Mds, JAX stock 011038) to obtain tamoxifen-inducible cardiomyocyte-specific deletion of Lmna (LmnaFlx / Flx:mcm). Flx / Flx We developed mice (mcm;Tg(Myh6-cre / Esr1)) in which Cre expression is driven by the mouse cardiac-specific alpha-myosin heavy chain promoter (αMHC or alpha-MHC;Myh6) that specifically express tamoxifen-inducible Cre recombinase (MerCreMer) in immature and adult cardiac myocytes. * )1Jmk, JAX stock 005657). The specificity of mcm Cre expression to cardiomyocytes was confirmed by crossing the Cre line to mT / mG reporter mice [MD Muzumdar et al., Genesis 45:593-605 (2007)] (Figure 1). 35). Sun1 - / - The generation of Lmna mice has been previously described [YHChi et al., Development 136:965-973 (2009)]. N195K / N195K Mice have also been described [LC Mounkes et al., Hum Mol Genet 14:2167-2180 (2005)]. - / - Because mice are sterile, Lmna Δ / Δ :Sun1 - / - and Lmna Flx / Flxmcm:Sun1 - / - The mice were the Sun1 Lamin-Cre mouse strains. + / - It was obtained by mating with mice.

[0168] To test for the insertion of the loxP site and the conditional deletion allele, genotyping was performed by a duplex PCR protocol using the following primers:

[0169] [ka]

[0170] To test for Sun1 deletion, the following primers were used:

[0171] [ka]

[0172] To test for the MyHC transgene, the following primers were used:

[0173] [ka]

[0174] To test for the presence of the mcm transgene, the following primers were used:

[0175] [ka]

[0176] Tamoxifen injection and tissue collection Young (14 days old) and adult mice (3-5 months old) received a single injection of 40 mg / kg tamoxifen (Sigma) dissolved in corn oil (Sigma). At various time points after tamoxifen injection, mice were either sacrificed by CO2 euthanasia or anesthetized using a gas mixture of 1.5% isoflurane (BioMac) and 1.5 L O2. Cardiac arrest was induced by injection of 15% KCl, followed by a PBS wash to remove blood. Hearts for paraffin embedding were further washed with 4% paraformaldehyde (PFA), left overnight in 4% PFA, dehydrated in 70% ethanol for at least 24 hours, and embedded in paraffin. Hearts for cryosectioning were embedded in gum tragacanth (Sigma), frozen in liquid N2-cooled isopentane (BDH-AnalaR), cut into 9 μm sections using a cryostat (Leica CM3050), collected onto charged slides, and stored at -20°C for histology and immunofluorescence staining. Hearts for protein and RNA extraction were snap-frozen in liquid N2 and stored for further processing.

[0177] Cardiomyocyte isolation Cardiomyocyte isolation was performed according to standard protocols [M. Ackers-Johnson et al., Circulation Research 119:909 (2016)]. Briefly, mice were anesthetized with isoflurane (0.5 L / min 100% O2, 4% isoflurane nebulizer dial). The mouse heart was arrested with 15% KCl, the descending aorta was isolated, and 7 mL of EDTA buffer was injected into the right ventricle to flush the heart. The ascending aorta was clamped using Reynolds forceps, and the whole heart was removed and placed in a 60 mm dish containing fresh EDTA buffer. The heart was digested by sequential injection of 10 mL of EDTA buffer, 3 mL of perfusion buffer, and 30–50 mL of collagenase buffer into the left ventricle. Using forceps, the digested heart was gently torn into smaller, approximately 1 mm pieces and subjected to gentle trituration. Enzyme activity was inhibited by adding 5 mL of stop buffer. The cell suspension was passed through a 100 μm filter and concentrated by four successive rounds of gravity sedimentation to finally obtain a highly pure myocyte fraction. The myocyte pellet was snap-frozen in liquid N2 and stored at −80°C for further processing.

[0178] Histological and immunofluorescence microscopy For histological studies, sections (9 μm) were stained with standard hematoxylin and eosin for cell morphology, Masson's trichrome stain to detect collagen, and TUNEL assay to detect apoptotic nuclei. Images were acquired with a Zeiss Axio Imager microscope. For immunofluorescence on frozen heart sections, sections were warmed to room temperature, rehydrated with PBS, blocked with MOM block (Vector Shields) and donkey serum (Sigma-Aldrich), and incubated with primary antibodies overnight at 4°C. Slides were then washed in PBS, incubated with secondary antibodies and Hoechst dye (Sigma-Aldrich) for 60 minutes, washed with PBS, and mounted with Prolong-Gold antifade reagent (Invitrogen). Primary antibodies were: LMNA / C N-18 (goat, 1:50, Santa Cruz), Sun1 monoclonal (mouse, undiluted, from B. Burke), PCM-1 (rabbit, 1:200, Sigma), and sarcomeric α-actinin (mouse, 1:100, Abcam); secondary antibodies were Alexa Fluor 488, 568, and 647 (1:250, Invitrogen). For immunofluorescence of isolated cardiomyocytes, myocytes were stained in suspension, gently spun down after each solution change, and then placed on glass slides for imaging using a Zeiss LSM510 inverted confocal microscope.

[0179] Western analysis of LMNA, SUN1, Ha tag, and GFP Whole hearts and quadriceps were homogenized in RIPA lysis buffer at 13,200 g and 4°C for 10 min. The cells were centrifuged for 1 minute. Total cell lysates were electrophoresed, transferred to PVDF membranes, and blocked with Odyssey blocking buffer (Li-Cor Biosciences). The membranes were incubated with primary antibodies for 2 hours at room temperature. The membranes were then washed in TBST wash solution and incubated in Odyssey IRDye secondary antibodies for 1 hour before visualization with an Odyssey infrared imaging system (Li-Cor Biosciences). Primary antibodies used for detection of LMNA / C were rabbit (Cell Signaling), specific for an epitope in the first 50 amino acids of LMNA, Sun1 monoclonal (mouse, 1:500, Burke), and control beta-tubulin (rabbit, 1:1000, Abcam).

[0180] Measurement of cardiac papillary muscle force Mouse papillary muscles from the left ventricle were prepared according to a previously described method [C.N. Toepfer et al., J Physiol 594:5237–5254 (2016)]. Briefly, the described mouse heart was immediately rinsed with ice-cold oxygenated Krebs-Henseleit solution containing 12 units / mL heparin sodium (EDQM) and 30 mM 2,3-butanedione monoxime (BDM, Sigma) to remove excess blood. The heart was then transferred to ice-cold Krebs-Henseleit solution in a glass Petri dish under a dissecting microscope equipped with a cooling stage. Cylindrical papillae (200–300 μm in diameter and 1.5–2 mm in length) were excised from the left ventricle. Perforated T-shaped aluminum clips were crimped onto both ends of the papilla preparation, and the prepared papilla mass was fixed using pins onto a glass Petri dish containing a layer of PDMS Sylgard 184 (Dow Corning). The teat preparation was immersed overnight in a 2% Triton X-100 solution at 4°C.

[0181] Force measurements were performed as previously described [C. Toepfer et al., J Biol Chem 288:13446-13454 (2013)]. T-shaped aluminum clips on both ends of the nipple preparation were attached to the hooks of a force transducer (AE801, HJK Sensoren+Systeme), and the servomotor of the experimental rig was glued with shellac in ethanol (Sigma) to minimize movement during the experiment. Nipple contraction force was measured at 20°C. Maximum contraction force was measured in an activating solution containing 32 μmol / L free Ca2+ (100 mM TES, 6.5 mM MgCl2, 25 mM Ca-EGTA, 5.7 mM Na2ATP, 20 mM glutathione, 21.5 mM sodium creatine phosphate, pH = 7.1, ionic strength 150 mmol / L). Data were collected and processed from the force transducer and a DAQ data acquisition system (National Instrument) using customized software programmed with LabVIEW 2013 (National Instrument). At least five fibers were tested in each mouse, with at least three mice tested per experimental group.

[0182] AAV9-DN-Sun1 and AAV9-GFP viruses The DN-Sun1 (SS-HA-Sun1L-KDEL) and GFP (SS-GFP-KDEL) vectors were prepared as previously described [M. Crisp et al., J Cell Biol. 172:41-53 (2006)]. Briefly, almost the entire luminal domain of Sun1 was tagged with HA at its NH2-terminus (HA-Sun1L). To deliver HA-Sun1L to the ER lumen and PNS as a soluble form, the human serum albumin signal sequence and signal peptidase cleavage site were fused to the NH2-terminus of HA-Sun1L, yielding SS-HA-Sun1L. To prevent its secretion, the KDEL tetrapeptide was fused to the COOH-terminus of SS-HA-Sun1L, forming the final SS-HA-Sun1L-KDEL vector. The HA-Sun1L region was replaced with a GFP sequence to generate SS-GFP-KDEL.

[0183] The DN-Sun1 and GFP fragments were amplified using the primers listed below (the same forward primer was used for both fragments), ligated into the pENN-AAV-cTnT-PI-eGFP plasmid (a gift from Dr. J. Jian), and digested with Ncol and Kpnl to generate Penn-AAV-cTnT-Sun1DN (Figure 10, sequence number 3).

[0184] [ka]

[0185] All restriction enzymes were purchased from NEB. PCR reactions were performed using Q5® Hot Start High-Fidelity 2X Master Mix (NEB, M0494L). Ligations were performed using NEBuilder® HiFi Isothermal assembly was performed using DNA Assembly Master Mix (NEB, E2621L). Primers used to construct the plasmids were ordered from IDT.

[0186] AAV virus was produced according to standard protocols [H. Wakimoto et al., in Current Protocols in Molecular Biology. (John Wiley & Sons, Inc., 2001)]. Materials provided by R. Foo: pAAV2 / 9—transplasmid encoding AAV replicase and capsid genes (SEQ ID NO: 2, available from the University of Pennsylvania Penn Vector Core); pAdDeltaF6—adenovirus helper plasmid (SEQ ID NO: 1) (available from the University of Pennsylvania Penn Vector Core); QIAGEN Plasmid Maxi Kit; HEK293T cells (ATCC); transfection reagent (polyethylenimine, e.g., Polysciences). AAV-DJ capsid was obtained from Cell Biolabs, Inc. pAAV2 / 9, AAV-DJ, pAdDeltaF6, DN-Sun1, and GFP plasmids were purified using a QIAGEN Plasmid Maxi Kit. HEK293T cells were transfected with a viral combination of pAAV2 / 9 plasmid, pAdDeltaF6 plasmid, and either DN-Sun1 or GFP plasmid. Cells were harvested, and the virus was purified by iodixanol gradient ultracentrifugation.

[0187] The following schedule was used for infection of mouse hearts. Mice were genotyped on postnatal day 10. Then, on postnatal day 14, mice received a single IP injection of Tmx (40 mg / kg mouse body weight), followed by intrathoracic injection of AAV9-DN-Sun1 or AAV9-GFP virus at a concentration of 5 x 10^10 vg / g on postnatal day 15. Adult mice (3-5 months old) received an IP injection of a single dose of Tmx (40 mg / kg mouse body weight), followed by intrathoracic injection of AAV at a concentration of 5 x 10^10 vg / g of AAV9-DN-Sun1 or AAV9-GFP virus. Young and adult mice were anesthetized using a gas mixture of 1.5% isoflurane (BioMac) and 1.5 L O2 before virus injection.

[0188] Plasmid construction and generation of Cas9 mRNA and sgRNA pX330 was obtained from Addgene (#42230, Cambridge, MA, USA). 20-nt Sun1 and Syne1 single guide RNA (sgRNA) sequences The gRNA was designed using the CRISPR Design Tool (crispr.genome-engineering.org). The region of the gene of interest was provided to the tool to identify suitable target sites. Because off-target mutations can occur during CRISPR / Cas9-mediated targeted mutagenesis in mice, the CRISPR Design Tool experimentally evaluates off-target genome modifications for each gRNA target site, provides computationally predicted off-target sites for each intended target, and ranks target sequences according to quantitative specificity analysis of the effects of base pair mismatch identification, location, and distribution. Complementary oligonucleotides containing the gRNA target sequence were annealed and cloned into the Bbsl site of pX330. The guide RNA sequence was as follows:

[0189] 5'-GCACAATAGCCTCGGATGTCG-3' (SEQ ID NO: 33) for Sun1ΔSUN 5'-CCGTTGGTATATCTGAGCAT-3' (SEQ ID NO: 34) for Syne1-stop 5'-GGTTATGGCCGATAGGTGCAT-3' (SEQ ID NO: 35) for tyrosinase 4a These plasmids (pSun1ΔSUN, pSyne1-stop, and pTyrosinase4a) were then sequenced to verify correct insertion of the target sequence. For in vitro transcription, PCR was performed using a general reverse primer (AAAAGCACCGACTCGGTGCC-3', SEQ ID NO: 36) and a gRNA-specific forward primer encoding the T7 promoter sequence as follows to generate appropriate transcription templates:

[0190] Sun1ΔSUN: 5′-TTAATACGACTCACTATAGCACAATAGCCTCGGATGTCG-3′ (SEQ ID NO: 37); Syne1-stop: 5'-TTAATACGACTCACTATAGCCGTTGGTATATCTGAGCAT-3' (SEQ ID NO: 38); Tyrosinase 4a: 5'-TTAATACGACTCACTATAGGTTATGGCCGATAGGTGCAT-3' (SEQ ID NO: 39) The gRNA PCR product was then subjected to agarose gel electrophoresis (1.5% agarose) to confirm successful PCR, gel purified, and used as a template for in vitro transcription using the MEGAshortscript T7 kit (Life Technologies). The gRNA was purified using the MEGAclear kit (Life Technologies) and eluted in RNase-free water. A sample of the purified gRNA was then subjected to agarose gel electrophoresis to confirm quality before injection into zygotes.

[0191] Generating mutant mice using CRISPR / Cas9 Three- to four-week-old C57BL / 6N females were superovulated with pregnant mare serum gonadotropin (Calbiochem, 36722, 5 IU / ml). 48 hours later, the females were injected with human chorionic gonadotropin (Sigma, CG10, 5 IU / ml) and mated with C57BL6 males. The following day, fertilized 0.5-dpc embryos were collected from the oviduct. Cas9 mRNA (Sigma, CAS9 mRNA, 100 ng / ul), tyrosinase 4a gRNA (50 ng / ul), and gene-specific gRNA (50 ng / ul) were co-injected into the cytoplasm of embryos in M2 medium (EmbryoMax®, Sigma) using a microinjection system (Nikon). Syne1-stop sgRNA was used to generate Syne1 C'T mutant mice, and Sun1ΔSUN sgRNA was used to generate Sun1ΔSUN mutant mice. The injected zygotes were cultured in KSOM (EmbryoMax® Sigma) containing amino acids for 2 hours in an incubator maintained at 37°C, 5% CO2 and 5% O2, and then transferred to 0.5 dpc pseudopregnant C3H-I were transplanted into CR females.

[0192] DNA extraction for genotyping of CRISPR / Cas9 mice Mouse tails were cut and placed in 1.5 ml Eppendorf tubes. 80 μl of lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 12) was dispensed into the tubes and heated at 95°C for 60 minutes. After heating, the buffer was neutralized with an equal volume of 40 mM Tris-HCl, pH 5. For certain applications, DNA was extracted and purified from mouse tails using the DNeasy Blood and Tissue Kit (QIAGEN).

[0193] Genotyping of CRISPR / Cas9 mice The genotypes of the CRISPR-modified mutant mice were identified by PCR followed by gel electrophoresis using high-resolution agarose (2% MetaPhor agarose, Lonza).

[0194] The primers for Syne1CT'Δ8 mice were: Forward: 5'-TGCTCCTGCTGCTGCTTATT-3' SEQ ID NO: 40, and Reverse: 5'-ACATGGTGGAGCATTTGTCTCC-3' SEQ ID NO: 41 It was.

[0195] Primers for Sun1 CRISPR mouse: Forward: 5'-TGACCTTGAGCTGAAACTGC-3' SEQ ID NO: 42, and Reverse: 5'-TCAGAACACTGGCACACACA-3' SEQ ID NO: 43 It was.

[0196] The genotype of Lmna mutant mice was identified as described in Example 1. To determine the sequence of the CRISPR-induced mutation, PCR products from mouse tail DNA were subjected to TOPO cloning (Zero Blunt™ TOPO™ PCR Cloning Kit, 450245, Thermo Fisher Scientific). Plasmid DNA from at least 10 bacterial colonies was isolated using a mini-prep kit (QIAGEN, QIAprepSpin, Miniprep Kit) and subjected to Sanger sequencing.

[0197] Obtaining myoblasts, fibroblasts, and cell cultures for CRISPR / Cas9 research To isolate myoblasts, limbs were obtained from euthanized mice, and the muscles were dissected from the bone. Tissue digestion was performed by incubating muscle tissue in an enzyme solution consisting of equal volumes of 2.4 U / ml Dispase II (Roche, cat. 04942078001) and 1% Collagenase II (GIBCO® Invitrogen, cat. 17101-015) in a 37°C water bath for 30 minutes, with occasional mixing every 10 minutes. After 30 minutes, the enzyme solution was neutralized in D10 medium (Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum). The mixture was then filtered through a 70 μm sterile filter (BD Falcon™, cat. 352350) and a 40 μm sterile filter (BD Falcon™, cat. 352340). The suspension was then centrifuged, the supernatant removed, and the cells were resuspended in F10 medium (GIBCO® Invitrogen, cat. 11550043) supplemented with 10 μg / ml bFGF (GIBCO®, cat. PHG0264) and plated onto 100 mm plates. After allowing the mouse adult fibroblasts to rest for 2-3 hours, the supernatant (containing floating myoblasts) was collected and coated with 0.15% gelatin (Sigma, cat. G1393). The cells were replated in 60 mm plates containing MAFs. D10 medium was added to the 100 mm plates containing MAFs. To differentiate the myoblasts into myotubes, the medium was changed to DMEM supplemented with 2% horse serum (Thermo Fisher Scientific GIBCO®, cat 16050122).

[0198] Immunoblotting for CRISPR / Cas9 research Whole cell lysates were generated using Lysis-M kit solution (cOmplete, Roche). Cells were washed in ice-cold PBS, lysed with Roche Lysis M buffer, and centrifuged at 14,000 g for 10 minutes to remove cell debris. To extract proteins from tissue samples, small slices of tissue were quickly placed in Lysing Matrix D tubes (MP Biomedicals) and flash-frozen in liquid nitrogen. After flash-freezing, the tubes were either stored at -80°C or used immediately for protein analysis. Protein extraction buffer (50 mM Tris (pH 7.4), 500 mM NaCl, 0.4% SDS, 5 mM EDTA (pH 7.4), 1× protease inhibitor (cOmplete™ EDTA-free protease inhibitor cocktail, catalog number 04693159001, Roche), 2% Triton, 1 mM dithiothreitol in distilled water) was added to the tissue, which was then homogenized using a FastPrep™-24 instrument (MP Biomedicals). Samples were then centrifuged at 14,000 g for 10 minutes to remove cellular debris. Protein concentrations were quantified using a bicinchoninic acid (BCA) protein kit (Bio-Rad), after which protein samples were loaded onto polyacrylamide gels to ensure equal amounts were analyzed. All protein samples were separated by SDS-PAGE gel analysis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) by wet transfer at 20V for 48 hours at 4°C. The membranes were blocked for 1 hour at room temperature in TBS containing 0.1% Tween 20 (TBST) supplemented with 5% dry milk (Anlene). Western blot analysis was performed using primary antibodies diluted in 5% dry milk (diluted in TBST). The membranes were incubated for 2 hours at room temperature or overnight at 4°C. For secondary antibodies, antibodies conjugated to horseradish peroxidase (HRP) (Invitrogen) were used for chemiluminescence imaging. The membranes were incubated with the secondary antibodies for 1 hour at room temperature. For immunoblots visualized by chemiluminescence, the membranes were incubated in ECL substrate (Pierce) for 1 minute, then exposed to chemiluminescence-sensitive film (Thermo Scientific) and subsequently processed.

[0199] Immunofluorescence for CRISPR / Cas9 research Cells were grown in 8-well slides (Ibidi) and fixed in ice-cold methanol at -20°C for 15 minutes. Cells were then rinsed twice in PBS and permeabilized and blocked with 0.1% Triton X and 3% BSA in PBS for 15 minutes at room temperature. Fixed and permeabilized cells were then rinsed three times in PBS. Samples were then incubated with primary antibodies (Table 2) for 2 hours at room temperature or overnight at 4°C. Samples were then washed three times with PBS and subsequently incubated with secondary antibodies (Life Technologies) and DAPI (Life Technologies) for 1 hour at room temperature. After three washes in PBS, cells were mounted with antifade agent (1% DABCO, 90% glycerol, 10% PBS) and examined using a Zeiss 510 Meta confocal microscope or an Axiovert 200 inverted epifluorescence microscope (Zeiss). Images were recorded and analyzed using Zeiss ZEN, Metamorph, or Image J (NIH) software.

[0200] [Table 2]

[0201] Mouse Genetics Lmna mice and tamoxifen injections were described in Example 1. Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 and Lmna Flx / Flxmcm :Syne1 C’TΔ8 / C’TΔ8 To obtain double mutant mice, Lmna Δ / + or Lmna Flx / Flxmcm Syne1 mice C’TΔ8 / C’TΔ8In the Syne2 mouse model, an IRES-β-gal neomycin selection cassette (PgkNeo) flanked by loxP sites was inserted into the Syne2 gene, resulting in the deletion of part of exon 102 and all of exons 103–104. The neomycin cassette was then removed by breeding with Cre recombinase-expressing mice. C’TΔ8 / + or Syne1 C’TΔ8 / C’TΔ8 Mouse Syne2 + / - or Syne2 - / - Mice carrying mutant Syne1 and Syne2 alleles were crossed with mice carrying mutant Syne1 and Syne2 alleles to obtain double mutant mice. Survival curves were plotted using the Kaplan-Meier method.

[0202] Human guide RNA sequences Potential guide RNA sequences that disrupt the human SYNE1 KASH domain or SUN1 SUN domain were determined using the CRISPR tool in Benchling software (Benchling Inc. USA) and are shown in Table 3.

[0203] [Table 3]

[0204] statistical analysis All statistical analyses were performed using Graphpad Prism software. Example 2 Cardiomyocyte-specific loss of Lmna leads to rapid onset of heart failure To further define the interaction between Sun1 and Lmna in postnatal pathology in mice, we developed a conditional Lmna gene that, when recombined by Cre activation, leads to the complete loss of lamin A / C protein. Flx / Flx By using a mouse strain (Figure 33), the Lmna gene was specifically ablated in different tissues [AS Wang et al., Differentiation; research in biological diversity, (2015); I. Solovei et al., Cell 152:584~598 (2013)]. lmna Flx / Flx Lmna mice by crossing them with Zp3-Cre mice Flx / Flx When Lmna was constitutively deleted in all tissues [W. de Vries et al., Genesis 26:110-112 (2000)], the mean postnatal survival time was 17.5 days (Fig. 26A). When the same deletion was induced in the absence of Sun1, Lmna Δ / Δ :Sun1 - / - Mice lived an average of 32.5 days, nearly doubling lifespan (Fig. 26A). The same Lmna deletion performed on a Sun2 null background resulted in Lmna Δ / Δ We found that loss of Sun1 did not extend the lifespan of mice, and that the lifespan extension was specific to loss of Sun1 (Figure 36). Because A-type lamins are widely expressed in almost all adult tissues, we next hypothesized that loss of Lmna, specifically in cardiomyocytes, would result in loss of Lmna. Δ / Δ We determined the extent to which Sun1 contributes to early postnatal death in mice. Furthermore, we wanted to determine whether loss of Sun1 could increase the lifespan of these mice, which contain Lmna-deficient cardiomyocytes. Flx / Flx These mice were crossed with a constitutive myh6Cre gene [R. Agah et al., J Clin Invest 100:169-179 (1997)] in which Cre expression is constitutive but restricted to cardiomyocytes and initiated during embryogenesis. Δ / Δ The survival time was slightly longer than that of Sun1 mice, averaging 26.5 days after birth (Fig. 26C). - / - When performed against a background mouse model, this resulted in a significant increase in lifespan up to and beyond 6 months of age (Figure 26C). To further define the loss of Lmna and its effects in postnatal / adult cardiomyocytes, Cre was induced by a single injection of tamoxifen (Tmx) [DS Sohal et al., Circ Res 89:20-25 (2001)]. Lmna carrying an inducible cardiomyocyte-specific Cre Tg(Myh6-cre / Esr1) (abbreviated here as mcm) was then cultured. Flx / FlxMice homozygous for the allele were obtained from this mating. Flx / Flx:mcm The mean survival time of mice after Cre induction was 27 days (Figure 27A). Controls were unaffected by Tmx injection. PCR and immunofluorescence analysis demonstrated that Lmna deletion resulted in the loss of Lmna Flx / Flx:mcm The results confirmed that the recombination was specific to cardiomyocytes, with no detectable recombination occurring in the brain, diaphragm, lung, liver, or skeletal muscle, nor in wild-type control animals (Figure 27B). Flx / Flx:mcm Mice exhibited labored breathing, a ruffled and ungroomed appearance of the fur, increased lethargy, and kyphosis (Figure 27C). Flx / Flx:mcm Immunofluorescence analysis of cardiac sections showed reduced levels of lamin A protein and cardiomyocyte nuclei lacking lamin A expression (Figure 27D). Lamin A protein levels were significantly elevated after Cre induction of Lmna Flx / Flx:mcm In the heart, Lmna did not induce Flx / Flx:mcm and Lmna + / + / mcm The Lmna expression level was 3.5-fold lower than that of the heart (Figure 27E). Flx / Flx:mcm By sampling mice, we estimated that it took 7–14 days for LMNA protein levels to fall by 50% after Cre induction (data not shown), a rate consistent with studies using siRNA LMNA knockdown in human fibroblasts [A. Buchwalter and M.W. Hetzer, Nature Communications 8:328 (2017); T. Sieprath et al., Nucleus 6:236–246 (2015)], which showed a further decline of 1.3-fold after 48 hours and 4-fold after 10.5 days. Echocardiograms (ECGs) performed 21 days after Cre induction showed that Lmna Flx / Flx:mcm In mice, Lmna Flx / Flx:mcm Compared to controls, the subjects revealed insufficient cardiac contractility (Fig. 28A). There was a significant decrease in ejection fraction (EF%) and fractional shortening (FS%) (P<0.0001) (Fig. 28B). Left ventricular systolic and diastolic internal diameters (LVID) were significantly decreased (Lmna Flx / Flx:mcm expanded compared to the control (Figure 28B).Flx / Flx:mcm Significantly fewer viable (brick-like) cardiomyocytes were detected in Lmna compared to controls. Flx / Flx:mcm +Tmx hearts (Figure 28C). Flx / Flx:mcm Cardiomyocytes isolated from +Tmx hearts contained large intracellular vacuoles This revealed that Lmna Flx / Flx:mcm Histological analysis of +Tmx hearts revealed infiltration of nucleated cells, and Lmna Flx / Flx:mcm Increased intercellular spaces between cardiomyocytes were evident compared to control hearts (Figure 28D). Flx / Flx:mcm The left ventricular cavity in the +Tmx heart was significantly enlarged, and simultaneously, Lmna Flx / Flx:mcm There was a significantly increased level of fibrosis in Lmna compared with controls (P = 0.0098). Flx / Flx:mcm +Tmx hearts (Figure 28D). Increased numbers of apoptotic cells compared to control hearts were also observed in Lmna Flx / Flx:mcm +Tmx hearts (Figure 28D). However, there was no evidence of detectable widespread DNA damage in cardiomyocytes, as assessed by Rad51, MRE11, H2AX phosphor-Ser, and 53BP1 immunostaining (data not shown).

[0205] Example 3 Sun1 deletion ameliorates cardiac pathology induced by Lmna loss Mice carrying the Lmna mutation exhibit a significant increase in lifespan and health in the absence of Sun1 [Chen et al., Cell 149:565-577 (2012)]. As described, inducible deletion of Lmna in cardiomyocytes (Lmna Flx / Flx:mcm +Tmx) resulted in death within one month after Cre induction (Figure 26C). Surprisingly, when the same deletion was induced on a Sun1 null background, mice survived for more than one year after Cre induction (Figure 26C). Flx / Flx:mcm Sun1 - / - Hearts from +Tmx mice were cultured at 3 weeks post-induction in Lmna Flx / Flx:mcm Sun1 + / +We determined the extent to which SUN1 loss ameliorated the pathological changes induced by Lmna loss in cardiomyocytes compared with hearts from +Tmx. Immunofluorescence imaging for lamin A / C identified many elongated and distorted nuclei. In some of these, residual lamin A / C was associated with Lmna loss. Flx / Flx:mcm Sun1 + / + +Tmx hearts migrated to one pole of the nucleus (Panel 1 and inset in Figure 29A). Flx / Flx:mcm Sun1 - / - In +Tmx hearts, even in the absence of lamin A / C staining, many elongated nuclei were present, but these showed little, if any, distortion (yellow arrowhead in panel 3 of Figure 29A). Western analysis of whole hearts revealed that Lmna Flx / Flx:mcm Sun1 - / - + In Tmx cardiac lysate, Lmna Flx / Flx:mcm Sun1 + / + A significant decrease in lamin A / C was observed compared to the control (P=0.0359) (Figure 29A, bottom panel). Flx / Flx:mcm Sun1 + / + +Tmx cardiomyocyte nuclei exhibited increased longitudinal length with a segmented appearance, with segments connected by narrow bridges (Figure 29A and C, panel 1, arrowheads). However, in the absence of Sun1, Lmna Flx / Flxmcm Sun1 - / - Cardiomyocyte nuclei showed no abnormalities or compartmentalization (Figure 29C, panels 3 and 4). Flx / Flx:mcm Sun1 - / - For less than 1% of cardiomyocytes from Flx / Flx:mcm Sun1 + / + 70% of the cardiomyocytes in the mice had ruptured or malformed nuclei (Figure 29C, panel 5).

[0206] A distinct enlargement of the left ventricle (LV) is called Lmna Flx / Flx:mcm Sun1 + / + Although it was clear in mice, Lmna Flx / Flx:mcm Sun1 - / - +Tmx hearts (Fig. 29B, panels 1 and 2). Flx / Flx:mcm Sun1 + / +The hearts exhibited significantly increased levels of fibrosis compared with controls (P<0.0001), whereas Lmna Flx / Flx:mcm Sun1 - / - There was no significant fibrosis in the heart (FIG. 29B, panels 3-5).

[0207] As a model of the left ventricular muscle's operating mechanism, we measured the force acting on the cardiac papillary muscle. Flx / Flx:mcm :Sun1 + / + + In Tmx papillary muscles, Lmna Flx / Flx:mcm Sun1 + / + +CTL significantly reduced by 66% (P=0.0028). In the absence of SUN1, Lmna Flx / Flx:mcm Sun1 - / - +Tmx cardiac papillary force was maintained at a level not significantly different from that of the control (Figure 29B, panel 6).

[0208] Echocardiograms performed before and after Cre induction were performed. Flx / Flx:mcm Sun1 + / + Lmna caused a progressive deterioration of cardiac contractility in +Tmx mice. Flx / Fl:mcm Sun1 - / - +Tmx mice (Fig. 29D). Flx / Flx:mcm Sun1 - / - In +Tmx mice, EF, FS, and longitudinal global strain (GLS) (GLS is an independent parameter used to assess myocardial contractility and is a better predictor of heart failure) were all significantly reduced in Lmna Flx / Flx:mcm Sun1 + / + +Tmx mice.

[0209] Aging Lmna 12-14 months after Tmx injection Flx / Flx:mcm Sun1 - / - PCR analysis of +Tmx hearts confirmed the persistent deletion of the Lmna gene (Figure 37C), and protein quantification revealed that Lmna expression was significantly elevated 12–14 months after TMX. Flx / Flx:mcm Sun1 - / -+ revealed a significant decrease in LMNA levels in Tmx hearts (Figure 37D). Flx / Flx:mcm Sun1 - / - Histological analysis of +Tmx hearts revealed no significant increase in fibrosis compared to controls (Figures 37A and B). However, echocardiograms of these aged mice showed no significant increase in fibrosis. Flx / Flx:mcm Sun1 + / + +CTL mice and Lmna Flx / Flxmcm Sun1 - / - +Tmx mice showed decreased EF and FS (Fig. 37E), whereas Lmna Flx / Flx The average survival time of the mice was 13-14 months (Figure 26C), and therefore the reduced contractile function was likely due to aging. Furthermore, these findings demonstrate that loss of Lmna in adult (2-3 month old) cardiomyocytes is sufficient to result in cardiac failure within 3-4 weeks after Cre activation, but this pathology is remarkably alleviated by deletion of Sun1, and this alleviation persists for 1 year.

[0210] Example 4 Loss of SUN1 extends the lifespan of Lmna missense mutants Because most cases of LMNA-induced DCM are caused by missense mutations, we determined the effect of loss of SUN1 on the lifespan and cardiac function of a previously described Lmna mutant mouse line (L.C. Mounkes et al., Hum Mol Genet 14:2167-2180 (2005)) that harbors the N195K missense mutation, which has been identified in two unrelated patients diagnosed with AD-EDMD (D. Fatkin et al., N Engl J Med 341:1715-1724 (1999); J.P. van Tintelen et al., Am Heart J 154:1130-1139 (2007)). Again, we found that the absence of SUN1 significantly extended the survival time of this mutant mouse line, along with improved cardiac function (Figure 26D). These findings were confirmed by obtaining mice heterozygous for the N195K mutation by introducing a loxP sequence into the WT-Lmna allele, i.e., Lmna N195K / Flx×Sun1 + / + In these mice, the Tmx-inducible cardiomyocyte Cre allele (Lmna) was introduced. N195K / Flx:mcm +Tmx) results in the deletion of the WT flox Lmna allele, causing cardiomyocytes to become Lmna N195K / - These mice had a mean survival time of less than 50 days and were hemizygous for the Lmna mutation. N195K / N195K The lifespan of Lmna mice was half that of homozygous mice (Figure 30A). N195K / Flx:mcm When the +Tmx mutation was induced on a Sun1-null background, lifespan was significantly extended from less than 50 days to more than 200 days (Figure 30A), revealing that loss of Sun1 was also effective in preventing DCM caused by Lmna missense mutations, specifically in cardiomyocytes.

[0211] Echocardiograms performed before and after Cre induction were performed. N195K / Flx:mcm Sun1 + / + Lmna causes progressive deterioration of cardiac contractility in mice N195K / Flx:mcm Sun1 - / - This was revealed by comparison with mice (Figure 30B). Loss of SUN1 significantly reduced the expression of Lmna N195K / -:mcm Sun1 - / - In mice, EF, FS, and longitudinal global strain (GLS) were all significantly reduced by Lmna. N195K / -:mcm Sun1 + / + Conserved compared to mice (Figure 30B).

[0212] Example 5 AAV9-mediated transduction and expression of DNSun1 Flx / Flx:mcm Extends survival of +Tmx mice These results demonstrate that genetically ablating SUN1 function or genetically reducing SUN1 levels may have therapeutic value in treating DCM. We next examined whether this was due to complete elimination of SUN1 function, overcoming toxic SUN1 overload without altering SUN1 levels and by specifically disrupting SUN1's LINC complex-associated role in linking the nucleus to cytoskeletal components by tethering KASH domain proteins in the ONM. To distinguish between these two possibilities, we used adenovirus-associated virus (AAV) to specifically transduce and express a dominant-negative SUN1 minigene [M. Crisp et al., J Cell Biol 172:41-53 (2006)] in cardiomyocytes, whose protein product can compete for both SUN1-KASH and SUN2-KASH binding in cardiomyocyte nuclear cisternae. The Sun1 gene was tagged at its N-terminus with an HA (HA-Sun1L) epitope in a region corresponding to the entire luminal domain. To localize the resulting protein product to the endoplasmic reticulum (ER) and nuclear cisternae (between the INM and ONM—PNS), the signal sequence and signal peptidase cleavage site of human serum albumin were fused to the N-terminus of HA-Sun1L to obtain SS-HA-Sun1L. To prevent secretion of the miniprotein, the KDEL tetrapeptide was linked to the C-terminus of SS-HA-Sun1L to form SS-HA-Sun1L-KDEL (Figure 34). The signal sequence may ensure that HA-Sun1KDEL accumulates within the continuous perinuclear ER and PNS lumen within the cell. The cDNA sequence encoding the minigene was fused to the chicken cardiotroponin promoter (cTnT) to ensure that the minigene was transcribed exclusively in cardiomyocytes [KM Prasad et al., Gene Ther 18:43-52 (2011)]. A diagram of how SS-HA-Sun1L (DN-Sun1) translocates KASH domain proteins from the LINC complex in the PNS to the ER is shown in Figure 15 (third panel) and Figure 31B.

[0213] To verify that DN-Sun1 functioned in cardiomyocytes (CMs), we first transduced human CMs derived from iPS cells using the AAV-DJ system (D. Grimm et al., J. Virol. 82(12):5887-911 (2008)), which provides a higher infection rate in cultured cells than the AAV9 serotype and is used to transduce DN-Sun1 under the transcriptional control of the cTnT promoter in mouse hearts. DN-Sun1 was effective in displacing Nesprin-1 from the nuclear envelope in CMs expressing DN-Sun1, as shown in Figure 31D. Cells expressing high and low levels of DN-Sun1 are indicated by gray and white arrowheads, respectively. High levels of DN-Sun1 expression resulted in the dislocation of Nesprin-1 from the nuclear envelope. This confirmed that DN-Sun1 was effective in disrupting the LINC complex in CMs.

[0214] AAV (serotype 9) was used to deliver the DN-Sun1 minigene into the heart of postnatal mice. Transduction and expression were achieved by intraluminal injection. The procedure is summarized in Figure 31A. All mice were sacrificed for analysis 100 days after Tmx injection. PCR detection of Lmna deletion in the heart confirmed Cre induction by Tmx injection (Figure 31C). To determine the localization and expression level of the DN-Sun1 minigene, total protein was extracted from half of the heart. Western analysis revealed strong expression of both AAV9-DNSun1 protein and AAV9-GFP control protein (injection dose: 5 × 10^10 vg / g mouse) 99 days after AAV injection (Figure 31C). The expression levels of both proteins depended on the dose of injected viral particles (Figure 38). Expression of either AAV9-DNSun1 protein or AAV9-GFP protein did not affect LMNA protein levels (Figure 39A). Immunofluorescence analysis revealed that at 5x10^10 vg / g AAV9-GFP, a greater percentage of cardiomyocytes expressed GFP compared with the levels resulting from a ten-fold lower dose of viral particles (5x10^9 AAV9-GFP) (Figures 39B and 39C).

[0215] Lmna injected with AAV9-GFP control Flx / Flx:mcm +Tmx mice lived an average of 34.5 days after Tmx induction, whereas Lmna mice injected with AA9-DNSun1 (5×10^10vg / g mouse) Flx / Flx:mcm +Tmx mice lived significantly longer, with the majority surviving at least 100 days post-Tmx before being sacrificed for analysis (P=0.0002). (Figure 20 shows the results for the early time period for male and female mice, and Figure 31E shows the results at 100 days with separate graphs for male and female mice, where mice with different virus injection titers were removed.) Echocardiography analysis showed that Lmna Flx / Flx:mcm +Tmx+AAV9-DNSun1 hearts were Lmna at 35 days after Tmx Flx / Flx:mcm The Lmna+Tmx+AAV9-GFP hearts functioned better than the Lmna+Tmx+AAV9-GFP hearts (Figure 31G). Flx / Flx:mcm +Tmx+AAV9-DNSun1 mice were alive at 100 days post-induction, but both EF% and FS% were significantly higher than those of the control Lmna Flx / FlxWT +Tmx mice (Fig. 31G). At 35 days after Tmx, increased fibrosis was observed in Lmna mice. Flx / Flx:mcm +Tmx+AAV9-DNSun1 Heart and Lmna Flxx / Flxxmcm +Tmx+AAV9-GFP were detected in both hearts (Fig. 31F), but Lmna Flx / Flx:mcm Fibrosis in the heart with +Tmx+AAV9-DNSun1 was associated with Lmna Flxx / Flxxmcm +Tmx+AAV9-GFP hearts (lower panel in Figure 31F).

[0216] Example 6 Disruption of the LINC complex in mice using CRISPR / Cas9 Mice harboring multiple Lmna mutations, both systemic and cardiac-specific, exhibit significantly increased lifespan and health in the absence of Sun1 [(C.Y. Chen et al., Cell 149:565-577 (2012) and Examples 2-4]. Prior to the findings described in Examples 2-5, the mechanism of this rescue was unclear but was speculated to be due to a toxic effect of excess Sun1 in Lmna mutants [Chen et al., Cell 149:565-577 (2012)]. AAV-mediated expression of a dominant-negative LINC complex-disrupting transgene ameliorates the pathology associated with Lmna mutations [Example 5]. The findings in Examples 2-5 are consistent with the idea that LINC complex function, rather than excess Sun1, is the molecular driver of Lmna pathology. This is supported by the finding that genetic disruption of the LINC complex through loss of Sun1 and Sun2 in mice [K. Lei et al., Proc Natl Acad Sci USA 106:10207-10212 (2009)] or cardiac-specific disruption of Nesprin-1 and Nesprin-2 [Banerjee et al., PLOS Genet 10(2):e1004114 (2014)] was surprising because it resulted in a variety of pathologies.

[0217] To develop an alternative method for disrupting the LINC complex in vivo, we investigated whether CRISPR / Cas9 genome editing could be used to disrupt the SUN and KASH domains of proteins that make up the LINC complex. Because both the SUN and KASH domains are located at the C-terminus of the respective proteins, we hypothesized that CRISPR guide RNAs targeting the 3' end of genes encoding SUN or KASH domain proteins might result in premature termination codons after CRISPR-induced non-homologous end joining. This could result in truncated proteins with mutated C-terminal SUN or KASH domains. While the truncated proteins could be expressed and membrane-localized, they would be unable to interact with their cognate LINC complex partners. In Example 2, we found that loss of Sun2 did not ameliorate Lmna-associated pathology. Therefore, we chose to target the Sun1 SUN domain using CRISPR because Sun1 is believed to be the dominant SUN domain protein mediating Lmna pathology. Among KASH domain proteins, only Nesprin-1, Nesprin-2, and Nesprin-3 are ubiquitously expressed [H.F. Horn, Current Topics in Developmental Biology 109:287-321 (2014)]. Nesprin-1 and Nesprin-2 are closely related paralogs with overlapping functions. They interact with the actin and microtubule cytoskeleton, whereas Nesprin-3 appears to interact specifically with intermediate filaments [Kim et al., Biol. Chem. 396:295-310 (2015)]. Because we already had Nesprin-2 and Nesprin-3 mutant mouse lines obtained by conventional gene targeting available in our laboratory, we chose to target the KASH domain of Nesprin-1 using CRISPR to test whether CRISPR / Cas9 could be used in vivo to treat laminopathies.

[0218] The Sun1 gene and the Syne1 gene, encoding the Nesprin-1 protein, were directly targeted in vivo by microinjecting Cas9 mRNA into C57 / B16 mouse zygotes with either a gRNA targeting the SUN1 domain (5'-GCACAATAGCCTCGGATGTCG-3', SEQ ID NO: 66) or a gRNA targeting the KASH1 domain (5'-CCGTTGGTATATCTGAGCAT-3', SEQ ID NO: 67), followed by transplantation into surrogate mothers. Note that the SUN1 gRNA targets Sun1, which is upstream of the SUN domain, removing it. Co-injection with a gRNA targeting the tyrosinase gene (5'-GGTTATGGCCGATAGGTGCAT-3', SEQ ID NO: 68) resulted in CRISPR-edited offspring with white or mosaic coat color due to tyrosinase disruption. These pups were genotyped to confirm successful gene disruption and used as founder animals to establish Sun1 or Nesprin-1 mutant colonies.

[0219] Characterization of mutant mice After Sanger sequencing of the founder animals and F1 progeny, we focused on characterizing a Sun1 mutant allele with a 7-bp deletion (Sun1_del7 or Sun1Δ7, SEQ ID NO: 71) and a 4-bp insertion (Sun1_plus4, SEQ ID NO: 70) (Figure 40A), as well as a Syne1 (Nesprin-1) mutant allele with an 8-bp deletion (Syne1_CTdel8 or Syne1C'TΔ8, SEQ ID NO: 76) (Figure 41A). The Sun1 mutant allele was predicted to produce an mRNA with a premature stop codon, resulting in a truncated Sun1 protein lacking the SUN domain (Figure 40B). Tail tip fibroblasts were isolated from Sun1 homozygous mutant animals. Immunofluorescence staining revealed loss of Sun1 protein (Figure 40C), suggesting that the CRISPR-generated indel triggered nonsense-mediated decay of Sun1 mRNA. It is unclear whether the location of the mutation, which was outside the SUN domain rather than inside it, had any effect on the expression of the mutant gene. Because we were unable to obtain Sun1 mutant alleles producing defective Sun1 protein and instead essentially obtained Sun1 null animals, we did not further characterize these mutant lines.

[0220] The Syne1 C'TΔ8 allele is predicted to produce a protein (Figure 41B, SEQ ID NO: 78) in which the last 11 amino acids in the wild-type sequence (SEQ ID NO: 77) are mutated, followed by an additional 50 amino acids encoded by an alternative reading frame. Immunoblotting performed on Syne1WT and Syne1C'TΔ8 heart and muscle tissues revealed a band of approximately 120 kDa corresponding to the Nesprin-1α isoform of the Syne1 gene, which is enriched in striated muscle in WT (Figure 41C, D). In C'TΔ8 heart and muscle tissues, the putative Nesprin-1α polypeptide appeared less abundant and had a lower electrophoretic mobility than in the wild-type (Figure 41C, D). This is consistent with the 8-bp deletion in the Syne1C'TΔ8 allele introducing a novel stop codon downstream, resulting in a higher molecular weight protein. In addition, a band of approximately 1 MDa, likely corresponding to Nesprin-1Giant, was observed in cardiac tissue from both Syne1WT and Syne1C'TΔ8 mice.

[0221] Immunofluorescence analysis of mouse adult fibroblasts (MAFs) derived from 12-week-old mice revealed that Nesprin-1 was mislocalized from the nuclear envelope to the cytoplasm in Syne1C'TΔ8 MAFs (Figure 4A). Similarly, in myotubes, Nesprin-1 redistributed to the cytoplasm in Syne1C'TΔ8 myotubes compared to Syne1WT myotubes (Figure 4B). Other LINC complex and NE proteins, such as SUN1, SUN2, emerin, and lamin A, remained localized to the NE (Figure 4C). Consistent with previous reports [Gimpel et al., Curr. Biol. 27:2999-3009.e9.(2017)], disruption of Nesprin-1 in myotubes resulted in the mislocalization of the centrosomal proteins PCM1, Pcnt, and Akap450 from the myotube nuclear envelope (Figure 4D). Mislocalization of Nesprin-1 from the nuclear envelope is consistent with disruption of the Nesprin-1 KASH domain, preventing the Nesprin-1C'TΔ8 mutant protein from interacting with the SUN domains of Sun1 and Sun2, which would normally restrict Nesprin-1 to the nuclear envelope. Because the transmembrane domain is not disrupted, Nesprin-1 is likely mislocalized to the endoplasmic reticulum (ER) in the C'TΔ8 mutant because the ER and nuclear cisternae form a continuous membrane system.

[0222] Similar to one previously reported Nesprin-1 mouse model [Zhang et al., Development 134(5):901-908 (2007)], and in contrast to two other models [Puckelwartz et al., Hum Mol Genet 18:607-620 (2009); Zhang et al., Hum Mol Genet 19:329-341 (2010)], the disrupted KASH domain of Nesprin-1 did not result in any obvious phenotypic differences between Syne1 wild-type (WT) and Syne1C'TΔ8 mutants (Figure 45A-B). Both male and female homozygous mutants were fertile, and there was no significant difference in body weight between Syne1WT and Syne1C'TΔ8 mice (Figure 45C). Syne1C'TΔ8 mice also did not exhibit any growth retardation or overt muscular dystrophy, and did not show any difficulties with movement or grooming, which could be indicators of muscle loss.

[0223] To explore the role of other KASH domain proteins in Lmna pathology, we generated mouse mutants for Syne2, which encodes Nesprin-2, by conventional gene targeting (Figure 46A). To characterize the mutations, we performed immunofluorescence microscopy of tail tip fibroblasts. Syne2 - / - Homozygous mutant fibroblasts expressed little or no Nesprin-2 (Fig. 46B). Consistent with previous findings [Zhang et al., Development 134(5):901-8(2007)], Sy ne2 - / - The mice were apparently normal and had neither growth retardation nor infertility, but the Nesprin-1 / 2 double mutant mice (Syne1 C’TΔ8 / C’TΔ8 :Syne2 - / - ) was perinatally lethal (Figure 46C).

[0224] Disruption of the Nesprin-1 KASH domain ameliorates Lmna pathology Even if Nesprin-1 is still expressed, the Nesprin-1-containing LINC complex is not involved in the expression of Syne1. C’TΔ8 / C’TΔ8Because AAV-mediated disruption of the LINC complex in vivo using dominant-negative SUN1 rescues Lmna pathology (Example 5), we reasoned that the "KASH-free" Nesprin-1 mutant allele we generated might also rescue Lmna pathology. To test this hypothesis, we created an Lmna null (Lmna Δ / Δ Mice heterozygous for the Lmna allele (Example 1) were crossed with Syne1C'TΔ8 mice to produce Lmna Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 We obtained double mutant mice. Δ / Δ The mice lived for 15–17 days, but Lmna Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 The double mutant mice lived for up to 42 days (Figure 24). C’TΔ8 Lmna null mice heterozygous for the allele did not experience any survival benefit. - / - Lmna on a homozygous mutant background Δ / Δ Mice also did not experience increased survival (FIG. 47), indicating that Lmna pathology is primarily mediated by the Nesprin-1 / Sun1 LINC complex.

[0225] Syne1 in mice with cardiac-specific loss of Lmna C’TΔ8 / C’TΔ8 To examine the effect of the allele, we transduced conditional Lmna mice carrying an inducible cardiomyocyte-specific Cre Tg(Myh6-cre / Esr1) (abbreviated as mcm here) in which Cre expression was induced by a single injection of tamoxifen (Tmx). Flx / Flx Mice homozygous for the allele were used as described in Examples 1 and 2. Cardiac-specific deletion of Lmna resulted in death within one month, whereas homozygous Syne1 C’TΔ8 / C’TΔ8 Mice with the same deletion induced on the background survived for at least 120 days after Tmx induction (FIG. 25, no change from day 80 to day 120).

[0226] Example 7 Methods for screening small molecules that block SUN-KASH interaction Crystallographic studies of human SUN2 reveal that the SUN domain assembles into a cloverleaf-like trimeric structure [Sosa et al., Cell 149(5):1035-47(2012)]. Trimerization is mediated by a triple-helical coiled-coil with an estimated length of 40-45 nm. This is sufficient to bridge the nuclear cisternae (PNS), allowing the SUN and KASH domains to directly interact [Sosa et al., Cell 149(5):1035-47(2012)]. The KASH-binding site is primarily formed within a groove formed at the interface between adjacent SUN domains (Figure 5B, left panel of Figure 21). This groove accommodates a portion of the approximately 18-residue KASH domain in an extended conformation. However, that portion is the C-terminal tetrapeptide of the KASH domain, which features three proline residues followed by a terminal aliphatic residue, Leu or Thr (for Nesp1 and Nesp2, respectively), and is crucial for SUN-KASH interaction (right panel of Figure 21, adapted from Figure 1 in Sosa et al., Cell 149(5):1035-47(2012)). The importance of this tetrapeptide is that it is located in a well-defined pocket formed within a single SUN monomer. Modification of this peptide by any means, including the addition of a single residue (Ala) at the C-terminus, completely eliminates SUN-KASH association across the entire SUN-KASH contact region (Sosa et al., Cell 149(5):1035-47(2012) and left panel of Figure 22). The conclusion is that stable binding of KASH domains requires 18-20 residues, but it is the C-terminal tetrapeptide that actually initiates binding. Therefore, the SUN monomer Blocking the tetrapeptide-binding pocket in vivo can disrupt the SUN-KASH association. In this disclosure, we describe an AAV-based gene therapy strategy that disrupts the endogenous SUN-KASH interaction as a treatment for laminopathies, including dilated cardiomyopathy. Alternatively, small molecules that block the SUN-KASH interaction at the SUN-binding pocket can disrupt the LINC complex and similarly treat laminopathies. There are a variety of standard methods for screening small molecule drugs in vitro.

[0227] In vitro screens can be constructed using recombinant SUN and KASH domains, or KASH peptides, whose production methods have been published [Sosa et al., Cell 149(5):1035-47 (2012)]. One such screen involves an assay technique similar to an enzyme-linked immunosorbent assay (right panel of Figure 22, equivalent to Lepourcelet et al., Cancer Cell. 5(1):91-102 (2004)). Recombinant SUN domains are immobilized on a solid surface, typically a 96-well plate, and then complexed with recombinant KASH domains linked to an enzyme capable of generating a colorimetric or chemiluminescent readout. One method for enabling this linkage is to synthesize biotinylated KASH peptides, which can then be linked to commercially available streptavidin-horseradish peroxidase (HRP) conjugates. Candidate compounds are obtained from appropriate suppliers and screened in vitro for their ability to inhibit KASH-SUN association. Compounds that cannot inhibit the SUN-KASH interaction will result in wells in which recombinant SUN binds to the enzyme-linked KASH domain. After a washing step and incubation with a colorimetric or chemiluminescent HRP substrate, the presence of the SUN-KASH interaction is detected in a standard plate reader. If the compound can inhibit the SUN-KASH interaction, the KASH domain will be removed after the washing step, and the enzyme reaction in the well will be reduced or absent.

[0228] Alternatively, fluorescence anisotropy or polarization can be used to screen for small molecule inhibitors of the SUN-KASH interaction in vitro [Lea, WA and Simeonov, A. Expert Opin Drug Discov 6:17-32 (2011)]. This assay also uses recombinant SUN and KASH domains. The KASH domains are fluorescently labeled; for example, chemically synthesized KASH peptides can be easily functionalized with fluorescein moieties. The fluorescence anisotropy of interacting KASH domains that interact with the SUN domain can be measured using standard instruments such as a plate reader. Because fluorescent KASH changes fluorescence anisotropy when not bound to SUN, small molecule inhibitors that disrupt the SUN-KASH interaction can be easily detected.

[0229] As is typical in drug screening processes, compounds that successfully pass the in vitro primary screen can then be subjected to a cell-based secondary screen (Figure 23). In this case, immunofluorescence microscopy can be used to identify compounds that can dissociate the LINC complex. This is manifested as dispersion of KASH components to the perinuclear endoplasmic reticulum, while the cognate SUN protein is retained at the inner nuclear membrane. This microscopy-based assay can be initially performed on HeLa cells. Active compounds are then evaluated in cultured cells from disease-related tissue, such as cardiac cells. Additional secondary screens may include whether identified compounds are able to rescue the growth defect in Lmna knockout cells. After hit-to-lead optimization of identified compounds using standard methods, compounds can be tested for Lmna dilated cardiomyopathy in a mouse model of laminopathies, such as those described herein. Lead efficacy can be assessed using survival time and echocardiograms of mutant mice to assess cardiac function, as described herein.

[0230] Consideration DCM caused by LMNA is considered progressive and often leads to premature death or heart transplantation [M. Pasotti et al., J Am Coll Cardiol 52:1250-1260 (2008); MR Taylor et al., J Am Coll Cardiol 41:771-780 (2003)]. By age 60, 55% of LMNA mutation carriers will die of cardiovascular failure or undergo heart transplantation, compared with 11% of patients with idiopathic cardiomyopathy. Attempts to ameliorate DCM by adjusting pacemakers are, at best, only temporarily beneficial. Therefore, the development of novel therapeutic approaches to treat DCM caused by LMNA mutations is necessary.

[0231] The majority of LMNA mutations that cause DCM are dominant-negative missense. Conventional gene therapy to repair each mutation can be extremely challenging, and removal of the mutant allele, leaving the patient hemizygous for the remaining normal WT allele, can also result in heart failure [G. Bonne et al., Nature Genetics 21:285-288 (1999)]. Various other avenues downstream of lamin genes are being explored for potential therapeutic intervention, including mTOR inhibition with rapamycin / rapalogs [J.C. Choi et al., Science Translational Medicine 4:144ra102 (2012); F.J. Ramos et al., Science Translational Medicine 4:144ra103 (2012)] and inhibition of the MEK1 / 2 kinase pathway [W. Wu et al., Circulation 123:53-61 (2011)]. Both measures resulted in improved ventricular function and increased lifespan (10-40%), although the magnitude and long-term efficacy were significantly less than those observed by us with loss of Sun1.

[0232] Although the molecular mechanisms underlying the diverse phenotypes of laminopathies remain poorly understood, two alternative hypotheses have been proposed to explain the tissue-specific pathology: the first, the "gene regulation hypothesis," proposes that LMNA mutation / loss disrupts the equilibrium of various molecular pathways due to mutations that alter their interactions with NE proteins and chromatin, and this disruption alters gene expression. Evidence supporting this hypothesis includes the AKT-MTOR pathway [JC Choi et al., Science Translational Medicine 4:144ra102(2012)], the WNT / β-catenin pathway [L. Hernandez et al., Dev Cell 19:413-425(2010); C. Le Dour et al., Hum Mol Genet 26:333-343(2017)], the TGF-β / Smad pathway [JH Van Berio et al., Hum Mol Genet 14:2839-2849(2005); T. V. Cohen et al., Hum Mol Genet 22:2852-2869(2013)], and the MAP kinase pathway [A. Brull et al., Front Physiol 9:1533 (2018)] and the ERK1 / 2-CTGF / CCN2 pathway [M. Chatziflangkeskou et al., Hum Mol Genet 25:2220-2233 (2016)]. Although these changes have been described, none of them clearly demonstrate whether these changes are secondary compensatory effects in diseased tissue. Because Sun1 protein, but not mRNA, is upregulated in laminopathies, Sun1 also fits into this category of disrupted expression levels, leading to the proposal that the laminopathic phenotype is caused by toxicity from excess Sun1 [C.Y. Chen et al., Cell 149:565-577 (2012)].

[0233] The second hypothesis suggested that Lmna loss or mutation leads to increased nuclear fragility. As a result, mechanical stress and tension transmitted from the cytoplasm to the NE via the LINC complexes cause damage to the NE [J. Lammerding et al., J Clin Invest 113:370-378 (2004)]. This hypothesis is similar to that proposed for Duchenne muscular dystrophy (DMD), in which loss of dystrophin increases the fragility of the muscle cell membrane, leading to muscle cell rupture and death when tension-stress is applied during muscle contraction [DJ Blake et al., Physiol Rev 82:291-329 (2002)]. When subjected to mechanical strain, Lmna mutant fibroblasts exhibit nuclear deformation, defective mechanotransduction, and reduced viability, as well as increased nuclear rupture compared to WT nuclei at low and moderate pressures [J. Lammerding et al., J Clin Invest 113:370-378 (2004); J. Lammerding et al., J Cell Biol 170:781-791 (2005); J. Lammerding et al., J Biol Chem 281:25768-25780 (2006)]. In contracting mouse cardiomyocytes, the mechanical stress and tension caused by 500-600 contractions per minute are transmitted to the NE via the LINC complex, resulting in nuclear distortion, damage, and eventual death / loss, as shown in Figures 28 and 29. Presumably, such forces cause significant damage to the fragile NE of Lmna null cardiomyocytes, resulting in CM death. If the tension-stress hypothesis is damaging to the NE, then uncoupling the LINC complex by disrupting SUN1 could reduce tension-stress on the CM nucleus and prevent CM cell death in mutant CM (Figure 32A-C). One caveat here is that complete disruption of the LINC complex, which can occur after overexpression of DN-Sun1, could potentially be detrimental rather than therapeutic. At the cellular level, multiple mechanical phenomena, including intracellular force transmission, cell polarization, and cell migration, were affected after disruption of the LINC complex with dominant-negative SUN and KASH constructs [Lombardi et al., J Biol Chem 286(30):26743-53(2011)]. In animal models, Sun1 / Sun2 [Lei et al., Proc Natl Acad Sci 106(25):10207-12(2009)] and Nesprin-1 / Nesprin-2 [Zhang et al., Development 134(5):901-8(2007)] double mutant mice experience perinatal lethality, and cardiac-specific disruption of the KASH domain of Nesprin-1 and Nesprin-2 using an embryonic cardiac Cre driver (Nkx2.5-Cre) results in early-onset cardiomyopathy [Banerjee et al., PLOS Genet 10(2):e1004114(2014)].

[0234] We attempted to distinguish between the tension-stress hypothesis and the expression level hypothesis in cardiomyocytes by using a DN-Sun1 construct that competes with endogenous Sun1 and Sun2 proteins for KASH domain binding, thereby disrupting LINC complex linkage without directly altering Sun1 levels (Figures 32D and 34). DN-Sun1 was delivered to CMs under the cTnT promoter using an AAV9 vector with high affinity for CMs [C. Zincarelli et al., Mol Ther 16:1073-1080 (2008)]. Our results demonstrated successful delivery of GFP into cardiomyocytes (Figure 31C) and strong expression of both control GFP and DN-Sun1 proteins (Figure 31C), the latter of which resulted in dispersion of the KASH domain protein from cardiomyocyte nuclei (Figure 31D). Surprisingly, AAV-DN-Sun1 not only ameliorated the pathology in mice with greatly reduced cardiac Lmna levels, but also had no discernible effect on cardiac health in wild-type mice that would be expected to similarly experience complete LINC complex disruption in the heart (Figures 31E and 31G), suggesting that an intact LINC complex may be required during embryonic development but not postnatally.

[0235] Additionally, we used CRISPR / Cas9 in mice to generate a Syne1 mutant allele (C'TΔ8) that results in a truncated Nesprin-1 protein with a disrupted, non-functional KASH domain. Mice lacking Lmna globally or in the heart have shortened lifespans, whereas Syne1C’TΔ8 / C’TΔ8 The presence of homozygous mutations is significant Loss of Sun1 or AAV-mediated disruption of the LINC complex in vivo with a dominant-negative transgene resulted in similar rescue of Lmna pathology (Examples 2-5), whereas Sun2 and Nesprin-2 mutations did not. Together, these data suggest that the LINC complex, composed of Sun1 and Nesprin-1, drives pathology in Lmna mutant cells and animals.

[0236] Several reports have been published on the use of AAV to deliver CRISPR / Cas components in vivo to treat diseases. Our results predict that AAV-mediated CRISPR / Cas delivery, such as CRISPR / Cas9 targeting the Nesprin-1 KASH domain in affected tissues, can be used to treat laminopathies, including dilated cardiomyopathy. For example, a cardiotropic AAV (e.g., AAV9) can be used to deliver a transgene cassette containing a cardiac-specific promoter (e.g., cTnT) driving Cas endonuclease enzyme expression and an appropriate promoter (e.g., U6) driving gRNA expression to treat LMNA DCM. Because the packaging capacity of AAV is limited to 4.7 kb, the smaller Cas9 (saCas9) derived from Staphylococcus aureus is considered preferable to the larger, more commonly used Streptococcus pyogenes Cas9. Alternatively, other CRISPR enzymes such as Cpf1 may be used, which are small enough for AAV packaging and have a protospacer adjacent motif (PAM) that is more prevalent than saCas9 [Zetsche, B. et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163, 759-771 (2015)].

[0237] The guide RNA can target the 3' region of the Nesprin-1 gene, which encodes the KASH domain (Table 3). While we targeted the region adjacent to the stop codon, in principle, any gene region encoding the KASH domain can be targeted, since CRISPR-generated indels are likely to result in frameshift mutations that disrupt the KASH domain. However, because the final four amino acids in the KASH domain are known to be absolutely required for SUN domain interaction and thus LINC complex formation [Sosa et al., Cell 149(5):1035-47 (2012)], it is prudent to select a gRNA near the stop codon, so that even indels that do not result in frameshifts can still mutate the relevant KASH amino acids required for SUN-KASH interaction. Furthermore, because the Syne1 gene encoding Nesprin-1 is very large and has multiple splice isoforms and alternative initiation sites, it should be noted that guide RNAs targeting outside the KASH domain may produce some mutant Nesprin-1 isoforms but may not disrupt the expression of other isoforms of the Nesprin-1 protein, including KASH-containing isoforms. This may result in a functional or partially functional Nesprin-1 / Sun1 LINC complex that may still be able to drive pathology in Lmna mutants. Δ / Δ :Syne2 - / - Mouse is Lmna Δ / Δ This CRISPR / Cas9 strategy may not be extended to the KASH domain of Nesprin-2, as it is phenotypically indistinguishable from mouse.

[0238] We did not further investigate the Sun1 mutant mice generated in this study because we essentially obtained the already well-characterized Sun1 null mice instead of mice with Sun1 lacking the SUN domain. We believe that inducing CRISPR mutations in Sun1 led to nonsense-mediated decay (NMD) of the Sun1 transcript. The appearance of a premature stop codon (PTC) 50–55 nucleotides upstream of the exon-exon junction triggers NMD [Popp, M. W. and Maquat, LE Cell 165:1319-1322 (2016)]. Furthermore, PTCs appearing in the middle of the transcript are more likely to cause NMD [Eberle et al., PLOS Biology 6:e92 (2008); Reber et al., MBoC 29:75-83 (2018)]. In the Sun1_plus4 mutant, the PTC is located more than 55 nucleotides upstream of the exon-exon junction, making it more likely to cause NMD. In the Sun1Δ7 mutant, the PTC is located within 50 nucleotides of the exon-exon junction. However, for both mutants, we targeted the PTC upstream of the sizable SUN domain, so the PTC is located approximately two-thirds of the length of the transcript, making it more likely that these PTCs also cause NMD. To specifically disrupt the SUN domain in Sun1 without inducing null mutations, a strategy similar to that used for Nesprin-1—guiding a guide RNA to the 3' end of the coding region of the transcript—can be employed (Table 3). Previous efforts demonstrated that mutation of the tyrosine residue at the C-terminus of SUN2 (Y707F) to phenylalanine disrupted KASH binding [Sosa et al., Cell 149(5):1035-47(2012)]. This critical tyrosine residue is conserved in SUN1 (Y812 in Uniprot E9PHI4) and is present in the final coding exon of the SUN1 transcript. Selecting a gRNA 5' proximal to the Y812 codon could generate an indel mutation that mutates Y812 and causes a frameshift mutation that could disrupt KASH binding. Because the gRNA would be present in the final coding exon, it is unlikely to trigger NMD. Therefore, it is possible to envision a CRISPR / Cas9-based strategy for treating laminopathies by targeting the critical residues in the SUN1 SUN domain that are required for KASH binding.AAV can be used to deliver CRISPR enzyme and gRNA that targets SUN1 to appropriate diseased tissue, such as heart.In this case, disabling SUN1 from KASH binding can ameliorate the harmful effects of Lmna mutation.

[0239] Based on these results, we propose that loss of or mutations within Lmna cause instability in CM nuclei due to loss or incorrect assembly of the nuclear lamina, rendering the nuclei more susceptible to tension / stress exerted by the contracting sarcomeres of the CM via the LINC complex. After mutation of the KASH domain, the untethered LINC complex exerts less tension on the CM nucleus, allowing lamin-deficient cardiomyocytes to survive.

[0240] These results provide an opportunity to use AAV-mediated delivery of DN-Sun, DN-KASH, or direct mutation of endogenous SUN or KASH proteins as a potential treatment for laminopathies-associated DCM in patients. The AAV system as a therapeutic delivery vehicle in patients has been established and approved by the FDA for treating some diseases. The AAV system is becoming more widely used due to multiple ongoing clinical trials, including in patients with cardiac disease. However, even though tension-stress may be a major cause of death in Lmna-deficient CMs, Lmna Δ / Δ :Sun - / - Because mice with LMNA DCM die at a younger age than mice lacking Lmna specifically in CM, disrupting SUN1 may not be effective in preventing LMNA mutation-induced cell death in skeletal muscle. It remains to be determined which muscle group (or other tissues lacking Lmna) results in early lethality. However, in most LMNA DCM patients, heart failure is the cause of death, and our results indicate that disrupting the LINC complex in CM may be effective in preventing heart failure long-term.

[0241] References

[0242] [Table 4-1]

[0243] [Table 4-2]

[0244] [Table 4-3]

[0245] [Table 4-4] Aspects of the invention [Embodiment 1] An isolated nucleic acid molecule, comprising an expression vector and a transgene, the transgene being operably linked to the expression vector, and wherein expression of the transgene in a transfected cell results in disruption of a nucleoskeletal-cytoskeletal linker (LINC) complex in the transfected cell. [Aspect 2] The nucleic acid molecule according to Aspect 1, wherein the expression vector is a heart- or cardiomyocyte-specific expression vector. [Embodiment 3] The nucleic acid molecule according to embodiment 1 or 2, wherein the expression vector comprises a cardiac or cardiac muscle cell-specific promoter. [Aspect 4] The nucleic acid molecule according to Aspect 3, wherein the expression vector comprises a cardiac or cardiomyocyte-specific promoter selected from the group consisting of cardiac troponin T promoter (cTnT), α-myosin heavy chain (α-MHC) promoter, and myosin light chain (MLC2v) promoter. [Embodiment 5] The nucleic acid molecule according to embodiment 3 or 4, wherein the cardiomyocyte-specific promoter is a chicken cardiac troponin T (cTnT) promoter. [Embodiment 6] The nucleic acid molecule of any of the preceding embodiments, wherein the expression vector is a viral expression vector. [Aspect 7] The nucleic acid molecule of Aspect 6, wherein the viral expression vector is selected from the group consisting of lentivirus, adenovirus, and adeno-associated virus (AAV). [Embodiment 8] The nucleic acid molecule of any of the preceding embodiments, wherein the adeno-associated virus expression vector (AAV) has / is cardiac tropism. [Embodiment 9] The nucleic acid molecule of any of the preceding embodiments, wherein the AAV vector is selected from the group consisting of AAV9 (serotype 9), AAV1 (serotype 1), AAV6 (serotype 6), AAV8 (serotype 8), AAV2i8, and AAV9.45. [Embodiment 10] The nucleic acid molecule of any of the preceding embodiments, wherein the AAV vector is AAV9 (serotype 9). [Embodiment 11] The nucleic acid molecule of any of the preceding embodiments, wherein the transgene comprises nucleic acid sequences for expressing the luminal domain of a SUN domain-containing protein, an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence. [Aspect 12] A nucleic acid molecule according to aspect 11, wherein the luminal domain of the SUN domain-containing protein comprises a coiled-coil domain and a SUN domain. [Aspect 13] A nucleic acid molecule according to aspect 11, wherein the coiled-coil domain is upstream of the SUN domain. [Embodiment 14] The nucleic acid molecule of any of the preceding embodiments, wherein the transgene further comprises nucleic acid sequences for expressing an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence. [Embodiment 15] The nucleic acid molecule of any of the preceding embodiments, wherein the transgene comprises an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence, as well as a nucleic acid sequence for expressing either the luminal domain or the SUN domain of a SUN domain-containing protein. [Aspect 16] A nucleic acid molecule according to any one of aspects 11 to 15, wherein the SUN domain protein is SUN1 or SUN2. [Embodiment 17] A nucleic acid molecule according to any one of embodiments 11 to 16, wherein the N-terminal signal sequence is derived from a secreted protein or a type I transmembrane protein. [Embodiment 18] The nucleic acid molecule of embodiment 17, wherein the secretory protein or type I transmembrane protein is selected from the group consisting of human serum albumin, proinsulin, transferrin receptor, EGF receptor, preproopiomelanocortin, pancreatic digestive enzymes (e.g., proteases, amylases, and lipases), endoplasmic reticulum luminal proteins, such as protein disulfide isomerase, GRP94, and combinations thereof. [Aspect 19] The nucleic acid molecule according to Aspect 18, wherein the N-terminal signal sequence is derived from human serum albumin. [Embodiment 20] A nucleic acid molecule according to any one of embodiments 11 to 19, wherein the N-terminal signal sequence is not present upstream of any other tag at its N-terminus. [Embodiment 21] The nucleic acid molecule according to any one of embodiments 11 to 20, wherein the signal peptidase cleavage site is derived from one of the group consisting of human serum albumin, proinsulin, transferrin receptor, EGF receptor, prepro-opiomelanocortin, pancreatic digestive enzymes (e.g., proteases, amylases, and lipases), endoplasmic reticulum lumen proteins such as protein disulfide isomerase, GRP94, and combinations thereof, or is one of the group. [Aspect 22] The nucleic acid molecule according to Aspect 21, wherein the signal peptidase cleavage site is derived from human serum albumin. [Aspect 23] A nucleic acid molecule according to any one of aspects 11 to 22, wherein the C-terminal targeting peptide sequence prevents secretion of the peptide expressed from the transgene according to any one of aspects 1 to 19. [Embodiment 24] A nucleic acid molecule according to any one of embodiments 11 to 23, wherein the C-terminal targeting peptide sequence is a KDEL sequence. [Embodiment 25] The nucleic acid molecule according to any one of embodiments 1 to 24, wherein the transgene further comprises an epitope tag. [Embodiment 26] A nucleic acid molecule according to embodiment 25, wherein the optional epitope tag is located at the N-terminus or anywhere in the nucleic acid molecule except downstream (after) the C-terminal targeting peptide sequence [e.g., KDEL], or anywhere in the nucleic acid molecule except upstream (before) the N-terminal signal sequence. [Embodiment 27] The nucleic acid molecule of embodiment 26, wherein the optional epitope tag is selected from the group consisting of cellulose binding domain (CBD), chloramphenicol acetyltransferase (CAT), dihydrofolate reductase (DHFR), one or more FLAG tags, glutathione S-transferase (GST), green fluorescent protein (GFP), hemagglutinin A (HA), histidine (His), herpes simplex virus (HSV), luciferase, maltose binding protein (MBP), c-Myc, protein A, protein G, streptavidin, T7, thioredoxin, V5, vesicular stomatitis virus glycoprotein (VSV-G), and combinations thereof. [Aspect 28] A nucleic acid molecule according to aspect 27, wherein the epitope tag is hemagglutinin A (HA). [Embodiment 29] The nucleic acid molecule of any of the preceding embodiments, wherein the vector is an adeno-associated virus vector (AAV) comprising a chicken cardiac troponin T promoter (cTnT) and a transgene of any of embodiments 1 to 28, wherein the luminal domain of the SUN domain-containing protein is derived from SUN1, the N-terminal signal sequence and signal peptidase cleavage site are each derived from human serum albumin, the C-terminal targeting peptide sequence is a KDEL sequence, and the transgene further comprises hemagglutinin (HA) as an N-terminal epitope tag. [Embodiment 30] The nucleic acid molecule of any of the preceding embodiments, wherein the vector is an adeno-associated virus vector (AAV) comprising a chicken cardiac troponin T promoter (cTnT) and a transgene of any of embodiments 1 to 28, wherein the luminal domain of the SUN domain-containing protein is derived from SUN2, the N-terminal signal sequence and signal peptidase cleavage site are each derived from human serum albumin, the C-terminal targeting peptide sequence is a KDEL sequence, and the transgene further comprises hemagglutinin (HA) as an N-terminal epitope tag. [Embodiment 31] A nucleic acid molecule according to any one of embodiments 1 to 10, wherein the transgene comprises a nucleic acid sequence for expressing a KASH domain and an N-terminal stabilizing polypeptide sequence. [Embodiment 32] The nucleic acid molecule of embodiment 31, wherein the KASH domain comprises a transmembrane domain and a SUN-interacting peptide. [Embodiment 33] A nucleic acid molecule according to embodiment 31 or 32, wherein the KASH domain is selected from the group consisting of KASH1 (derived from Nesprin-1 (SYNE1 gene)), KASH2 (derived from Nesprin-2 (SYNE2 gene)), KASH3 (derived from Nesprin-3 (SYNE3 gene)), KASH4 (derived from Nesprin-4 (SYNE4 gene)), and KASH5 (derived from KASH5 / CCDC155 (KASH5 gene)). [Embodiment 34] A nucleic acid molecule according to any one of embodiments 1 to 10, wherein the transgene comprises a nucleic acid sequence for expressing a CRISPR-Cas or other synthetic nuclease system that modifies a nucleic acid encoding the SUN domain of an endogenous Sun protein or the KASH domain of an endogenous Nesprin protein. [Aspect 35] The nucleic acid molecule of aspect 34, wherein the transgene comprises a nucleic acid sequence for expressing CRISPR-Cas. [Aspect 36] A nucleic acid molecule according to any one of aspects 1 to 33, wherein the introduced gene is a dominant-negative construct. [Embodiment 37] The nucleic acid molecule of any of the preceding embodiments, wherein the transgene is a humanized or human transgene. [Embodiment 38] A nucleic acid molecule according to any of the preceding embodiments, wherein expression of the introduced gene results in disruption of a protein-protein interaction between SUN and KASH of the LINC complex. [Embodiment 39] A nucleic acid molecule described in embodiment 38, wherein the disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between proteins selected from the group consisting of Sun1+Nesprin-1, Sun2+Nesprin-1, Sun1+Nesprin-2, Sun1+Nesprin-3, Sun2+Nesprin-2, and Sun2+Nesprin-3. [Aspect 40] A nucleic acid molecule according to Aspect 39, wherein disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between Sun1 and Nesprin-1. [Embodiment 41] The nucleic acid molecule of any of the preceding embodiments, wherein the AAV vector is formulated for delivery to the myocardium of a subject. [Embodiment 42] A nucleic acid molecule of any of the preceding embodiments for use in treating a disease caused by one or more Lmna mutations in a subject. [Embodiment 43] The nucleic acid molecule of embodiment 42, wherein the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), acromangial dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and a disease set out in regular font in Table 1. [Embodiment 44] A nucleic acid molecule according to any of the preceding embodiments for use in treating cardiovascular disease in a subject. [Embodiment 45] A nucleic acid molecule according to embodiment 42 or 43, wherein the disease or cardiovascular condition is characterized by the presence of at least one Lmna mutation. [Embodiment 46] The nucleic acid molecule of embodiment 44 or 45, wherein the cardiovascular disease is selected from the group consisting of laminopathies, cardiomyopathies, such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, and isolated atrial fibrillation; muscular dystrophy (often associated with cardiomyopathy), such as cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, and cardiomyopathy associated with congenital muscular dystrophies; premature aging syndromes (which are primarily vascular, but may also have cardiac involvement), such as cardiomyopathy associated with atypical Werner syndrome and cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome, and the like, and the diseases shown in bold font in Table 1. [Aspect 47] An adeno-associated virus vector (AAV) comprising a cardiac troponin T promoter (cTnT) and a transgene according to any one of aspects 11 to 30 or 34 to 38. [Aspect 48] A pharmaceutical composition comprising a nucleic acid molecule according to any one of aspects 1 to 41. [Aspect 49] A method for treating a disease in a subject, the method comprising administering a pharmaceutically effective amount of a nucleic acid molecule according to any one of aspects 1 to 41 or a pharmaceutical composition according to aspect 48. [Aspect 50] The method of aspect 49, wherein the disease is characterized by the presence of at least one Lmna mutation. [Embodiment 51] The method of embodiment 49 or 50, wherein the Lmna mutation affects the lamin A isoform or the lamin C isoform, or both the lamin A and C isoforms, of the Lmna gene. [Aspect 52] A method according to any one of aspects 49 to 51, wherein the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), acromandibular dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and diseases listed in normal font in Table 1. [Embodiment 53] The method of any one of embodiments 49 to 51, wherein the disease is a cardiovascular disease selected from the group consisting of laminopathies, cardiomyopathies, such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, and isolated atrial fibrillation; muscular dystrophy (often associated with cardiomyopathy), such as cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, and cardiomyopathy associated with congenital muscular dystrophy; premature aging syndromes (which are primarily vascular, but may also have cardiac involvement), such as cardiomyopathy associated with atypical Werner syndrome and cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome; and the diseases shown in bold font in Table 1. [Aspect 54] A method according to any one of aspects 49 to 53, wherein the subject is a non-human mammal or a human. [Aspect 55] Use of a pharmaceutical composition according to aspect 48 or a nucleic acid molecule according to any one of aspects 1 to 41 in the manufacture of a medicament for treating a disease caused by one or more Lmna mutations or a cardiovascular disease. [Aspect 56] The use described in Aspect 55, wherein the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), acromandibular dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and the diseases shown in regular font in Table 1. [Embodiment 57] The use of embodiment 55, wherein the cardiovascular disease is selected from the group consisting of laminopathies, cardiomyopathies, such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-grade AV block and arrhythmias, and isolated atrial fibrillation; muscular dystrophies (often associated with cardiomyopathy), such as cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, and cardiomyopathy associated with congenital muscular dystrophies; premature aging syndromes (which are primarily vascular, but may also have cardiac involvement), such as cardiomyopathy associated with atypical Werner syndrome and cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome, as well as diseases shown in bold font in Table 1. [Aspect 58] A pharmaceutical composition according to aspect 48 for use in therapy. [Embodiment 59] A method for screening drug candidates capable of inhibiting the interaction of proteins of a LINC complex in a cell, comprising: (a) combining proteins of the LINC complex to form a first complex in the presence of a drug; (b) combining the proteins to form a second complex in the absence of the drug; (c) measuring the amount of the first complex and the second complex; and (d) comparing the amount of the first complex with the amount of the second complex. wherein if the amount of the first complex is less than the amount of the second complex, the drug is a drug candidate for inhibiting the interaction of proteins of the LINC complex in a cell. [Embodiment 60] The method of embodiment 59, wherein the drug candidate disrupts the protein-protein interaction between SUN and KASH of the LINC complex. [Aspect 61] The method described in aspect 60, wherein the drug candidate disrupts the interaction between Sun1 protein and Nesprin-1 protein. [Aspect 62] A method according to any one of aspects 59 to 61, wherein the screening is an in vitro screening. [Embodiment 63] A method according to any one of embodiments 59 to 62, wherein the complex is measured by ELISA or fluorescence anisotropy measurement. [Aspect 64] A method described in any one of aspects 59 to 63, wherein if the amount of the first complex is less than the amount of the second complex, the drug is a drug candidate for inhibiting the interaction of the proteins. [Embodiment 65] A method according to any one of embodiments 59 to 64, wherein recombinant SUN and KASH domains are used.

Claims

1. 1. A pharmaceutical composition for treating a disease caused by a mutation in LMNA, comprising a nucleic acid molecule comprising an adeno-associated virus expression vector (AAV vector), wherein the AAV vector comprises an operably linked transgene, wherein expression of the transgene in a transfected cell results in disruption of the nucleoskeletal-cytoskeletal linker (LINC) complex in the transfected cell, and wherein the transgene (i) comprises a nucleic acid sequence for expressing the SUN domain of SUN1, an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence that prevents secretion of the peptide expressed from the transgene, wherein expression of the transgene generates a dominant-negative SUN1; or (ii) a pharmaceutical composition comprising a nucleic acid sequence for expressing the KASH domain of Nesprin-1 and an N-terminal stabilizing polypeptide sequence, wherein expression of the transgene generates a dominant-negative Nesprin-1.

2. 2. The pharmaceutical composition of claim 1, wherein expression of the transgene results in disruption of the protein-protein interaction between SUN and KASH of the LINC complex.

3. The pharmaceutical composition of claim 1 or 2, wherein the transgene contains a nucleic acid sequence for expressing the SUN domain of SUN1, an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence that prevents secretion of the peptide expressed from the transgene, and the transgene contains a nucleic acid sequence for expressing the luminal domain of SUN1, and the luminal domain contains the coiled-coil domain and SUN domain of SUN1.

4. 4. The pharmaceutical composition of claim 1, wherein the C-terminal targeting peptide sequence is a KDEL sequence.

5. 3. The pharmaceutical composition according to claim 1, wherein the KASH domain of Nesprin-1 comprises the transmembrane domain of Nesprin-1 and a SUN-interacting peptide.

6. The pharmaceutical composition according to claim 1 , wherein the AAV vector is a heart- or cardiomyocyte-specific expression vector.

7. 7. The pharmaceutical composition of claim 1, wherein the AAV vector is selected from the group consisting of AAV9, AAV1, AAV6, AAV8, AAV2i8, and AAV9.

45.

8. The pharmaceutical composition of claim 1 , wherein the AAV vector comprises a cardiac or cardiomyocyte-specific promoter.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the AAV vector comprises a cardiac or cardiomyocyte-specific promoter selected from the group consisting of cardiac troponin T promoter (cTnT), α-myosin heavy chain (α-MHC) promoter, and myosin light chain (MLC2v) promoter.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the disease is selected from the group consisting of cardiovascular disease; restrictive skin disorder; familial partial lipodystrophy; mandibular acrodysplasia with lipodystrophy type A: metabolic syndrome; Charcot-Marie-Tooth disease type 2; Charcot-Marie-Tooth disease type 2B1; acroprogeria, Gottron type; arthropathy syndrome, autosomal recessive; non-insulin dependent diabetes mellitus; distal acroosteolysis, poikiloderma and joint stiffness; distal motor neuropathy; dropped neck syndrome; familial partial lipodystrophy (Dunnigan type); familial partial lipodystrophy (Keberling); generalized lipodystrophy syndrome; Harlerman-Stryff syndrome; cardio-hand syndrome, Slovenian type; and type A insulin resistance syndrome.

11. Cardiovascular diseases include laminopathies; cardiomyopathy; dilated cardiomyopathy (DCM); dilated cardiomyopathy 1A; dilated cardiomyopathy with conduction system disorders; cardiomyopathy with high-grade AV block and arrhythmias; isolated atrial fibrillation; muscular dystrophy; cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant); cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive); cardiomyopathy associated with limb-girdle muscular dystrophy type 1B; cardiomyopathy associated with congenital muscular dystrophy; premature aging syndrome; cardiomyopathy associated with atypical Werner syndrome; cardiomyopathy associated with Hutchinson-Gilford progeria syndrome; arrhythmogenic cardiomyopathy; arrhythmogenic right ventricular cardiomyopathy; atrial fibrillation; atypical Werner syndrome; atypical atrial fibrillation 11. The pharmaceutical composition of claim 10, wherein the compound is selected from the group consisting of: geriatric syndrome; autosomal dominant spinal muscular dystrophy; axonal neuropathy, muscular dystrophy, heart disease; axonal neuropathy, muscular dystrophy, heart disease, leukonychia; cardiac arrhythmia; cardiac conduction disorder; congenital fiber inequality; congenital muscular dystrophy; dilated cardiomyopathy with conduction system disorder; Emery-Dreifuss muscular dystrophy, autosomal dominant; Emery-Dreifuss muscular dystrophy, autosomal recessive; lamin-related ankylosing spinal muscular dystrophy; limb-girdle muscular dystrophy type 1B; isolated atrial fibrillation; muscular dystrophy; muscular dystrophy and lipodystrophy; neonatal progeria syndrome; spinal muscular atrophy with cardiac involvement; and sudden cardiac death.

Images